WO2000010588A2 - Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r - Google Patents
Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r Download PDFInfo
- Publication number
- WO2000010588A2 WO2000010588A2 PCT/US1999/018696 US9918696W WO0010588A2 WO 2000010588 A2 WO2000010588 A2 WO 2000010588A2 US 9918696 W US9918696 W US 9918696W WO 0010588 A2 WO0010588 A2 WO 0010588A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- egf
- cells
- antagonist
- goblet
- antibody
- Prior art date
Links
- 108060006698 EGF receptor Proteins 0.000 title claims abstract description 357
- 102000001301 EGF receptor Human genes 0.000 title claims abstract description 354
- 210000003097 mucus Anatomy 0.000 title claims abstract description 73
- 210000004072 lung Anatomy 0.000 title claims abstract description 24
- 229940044551 receptor antagonist Drugs 0.000 title 1
- 239000002464 receptor antagonist Substances 0.000 title 1
- 210000002175 goblet cell Anatomy 0.000 claims abstract description 192
- 239000005557 antagonist Substances 0.000 claims abstract description 71
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 42
- 230000004663 cell proliferation Effects 0.000 claims abstract description 28
- 239000007924 injection Substances 0.000 claims abstract description 12
- 238000002347 injection Methods 0.000 claims abstract description 12
- 238000012216 screening Methods 0.000 claims abstract description 8
- 241000700159 Rattus Species 0.000 claims description 73
- 238000000034 method Methods 0.000 claims description 71
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 66
- 108090000623 proteins and genes Proteins 0.000 claims description 52
- 241001465754 Metazoa Species 0.000 claims description 47
- 239000000203 mixture Substances 0.000 claims description 43
- 102000004169 proteins and genes Human genes 0.000 claims description 38
- 238000009472 formulation Methods 0.000 claims description 36
- 230000000692 anti-sense effect Effects 0.000 claims description 28
- 238000011282 treatment Methods 0.000 claims description 27
- 230000028327 secretion Effects 0.000 claims description 22
- 208000006673 asthma Diseases 0.000 claims description 20
- 230000005764 inhibitory process Effects 0.000 claims description 19
- 230000001225 therapeutic effect Effects 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- 210000002919 epithelial cell Anatomy 0.000 claims description 17
- 208000019693 Lung disease Diseases 0.000 claims description 14
- 238000000338 in vitro Methods 0.000 claims description 13
- 238000001727 in vivo Methods 0.000 claims description 12
- 238000010874 in vitro model Methods 0.000 claims description 11
- 238000010171 animal model Methods 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 6
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 5
- 229940043355 kinase inhibitor Drugs 0.000 claims description 5
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 4
- 230000007423 decrease Effects 0.000 claims description 4
- 229940116977 epidermal growth factor Drugs 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 239000003757 phosphotransferase inhibitor Substances 0.000 claims description 4
- 230000004936 stimulating effect Effects 0.000 claims description 4
- 241000700199 Cavia porcellus Species 0.000 claims description 2
- 239000003963 antioxidant agent Substances 0.000 claims description 2
- 235000006708 antioxidants Nutrition 0.000 claims description 2
- AIBRSVLEQRWAEG-UHFFFAOYSA-N 3,9-bis(2,4-ditert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane Chemical group CC(C)(C)C1=CC(C(C)(C)C)=CC=C1OP1OCC2(COP(OC=3C(=CC(=CC=3)C(C)(C)C)C(C)(C)C)OC2)CO1 AIBRSVLEQRWAEG-UHFFFAOYSA-N 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 claims 1
- 230000003078 antioxidant effect Effects 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 45
- 230000002685 pulmonary effect Effects 0.000 abstract description 11
- 238000003556 assay Methods 0.000 abstract description 8
- 230000003843 mucus production Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 155
- 210000000440 neutrophil Anatomy 0.000 description 100
- 101000972282 Homo sapiens Mucin-5AC Proteins 0.000 description 88
- 230000014509 gene expression Effects 0.000 description 88
- 102100022496 Mucin-5AC Human genes 0.000 description 75
- 229920000936 Agarose Polymers 0.000 description 72
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 71
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 71
- 210000000981 epithelium Anatomy 0.000 description 67
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 64
- 238000010186 staining Methods 0.000 description 61
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 54
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 54
- 108010063954 Mucins Proteins 0.000 description 51
- 230000000694 effects Effects 0.000 description 51
- 238000004519 manufacturing process Methods 0.000 description 46
- 102000015728 Mucins Human genes 0.000 description 43
- 230000004913 activation Effects 0.000 description 43
- 238000003786 synthesis reaction Methods 0.000 description 41
- 239000006228 supernatant Substances 0.000 description 36
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 36
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 33
- 235000019504 cigarettes Nutrition 0.000 description 32
- 206010054949 Metaplasia Diseases 0.000 description 31
- PRQROPMIIGLWRP-UHFFFAOYSA-N N-formyl-methionyl-leucyl-phenylalanin Chemical compound CSCCC(NC=O)C(=O)NC(CC(C)C)C(=O)NC(C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-UHFFFAOYSA-N 0.000 description 31
- 230000015689 metaplastic ossification Effects 0.000 description 31
- 201000010099 disease Diseases 0.000 description 28
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 28
- 239000000779 smoke Substances 0.000 description 28
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 26
- 239000002953 phosphate buffered saline Substances 0.000 description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 24
- 108010058846 Ovalbumin Proteins 0.000 description 23
- 230000027455 binding Effects 0.000 description 23
- 150000001875 compounds Chemical class 0.000 description 23
- 229940092253 ovalbumin Drugs 0.000 description 23
- 239000003112 inhibitor Substances 0.000 description 22
- 101000972276 Homo sapiens Mucin-5B Proteins 0.000 description 21
- 210000002469 basement membrane Anatomy 0.000 description 21
- 238000006366 phosphorylation reaction Methods 0.000 description 19
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 19
- 101150031922 MUC5AC gene Proteins 0.000 description 18
- 238000011534 incubation Methods 0.000 description 18
- 230000026731 phosphorylation Effects 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 18
- 239000003446 ligand Substances 0.000 description 17
- 230000007246 mechanism Effects 0.000 description 17
- 239000000443 aerosol Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- 210000003437 trachea Anatomy 0.000 description 16
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 15
- 229960004397 cyclophosphamide Drugs 0.000 description 15
- 208000024891 symptom Diseases 0.000 description 15
- 239000002299 complementary DNA Substances 0.000 description 14
- 239000012528 membrane Substances 0.000 description 14
- 210000002955 secretory cell Anatomy 0.000 description 14
- 230000000638 stimulation Effects 0.000 description 14
- 238000002560 therapeutic procedure Methods 0.000 description 14
- 239000013598 vector Substances 0.000 description 14
- 206010020718 hyperplasia Diseases 0.000 description 13
- 108020004999 messenger RNA Proteins 0.000 description 13
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 229930040373 Paraformaldehyde Natural products 0.000 description 12
- 210000000621 bronchi Anatomy 0.000 description 12
- 239000003814 drug Substances 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 239000002502 liposome Substances 0.000 description 12
- 229940051875 mucins Drugs 0.000 description 12
- 230000003472 neutralizing effect Effects 0.000 description 12
- 229920002866 paraformaldehyde Polymers 0.000 description 12
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 12
- 210000000254 ciliated cell Anatomy 0.000 description 11
- 210000004379 membrane Anatomy 0.000 description 11
- 210000002850 nasal mucosa Anatomy 0.000 description 11
- 201000003883 Cystic fibrosis Diseases 0.000 description 10
- -1 DMTU Substances 0.000 description 10
- 230000003247 decreasing effect Effects 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 239000008187 granular material Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 230000001419 dependent effect Effects 0.000 description 9
- 230000008378 epithelial damage Effects 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 150000003254 radicals Chemical class 0.000 description 9
- 230000007115 recruitment Effects 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 210000000805 cytoplasm Anatomy 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000014616 translation Effects 0.000 description 8
- 206010061218 Inflammation Diseases 0.000 description 7
- 206010036790 Productive cough Diseases 0.000 description 7
- 208000024716 acute asthma Diseases 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 230000006378 damage Effects 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 238000007901 in situ hybridization Methods 0.000 description 7
- 230000004054 inflammatory process Effects 0.000 description 7
- 238000007912 intraperitoneal administration Methods 0.000 description 7
- 210000004940 nucleus Anatomy 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 208000023504 respiratory system disease Diseases 0.000 description 7
- 150000003384 small molecules Chemical class 0.000 description 7
- 210000003802 sputum Anatomy 0.000 description 7
- 208000024794 sputum Diseases 0.000 description 7
- 108090001007 Interleukin-8 Proteins 0.000 description 6
- 206010070834 Sensitisation Diseases 0.000 description 6
- 230000002159 abnormal effect Effects 0.000 description 6
- 210000000270 basal cell Anatomy 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- KDXZREBVGAGZHS-UHFFFAOYSA-M methohexital sodium Chemical compound [Na+].CCC#CC(C)C1(CC=C)C(=O)N=C([O-])N(C)C1=O KDXZREBVGAGZHS-UHFFFAOYSA-M 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- 230000000750 progressive effect Effects 0.000 description 6
- 239000003380 propellant Substances 0.000 description 6
- 238000001243 protein synthesis Methods 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000008313 sensitization Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- SEHPSBSPFWAVPK-NRFANRHFSA-N (2s)-2-[(2,7-dimethyl-9h-fluoren-9-yl)methoxycarbonylamino]-4-methylpentanoic acid Chemical compound C1=C(C)C=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC(C)=CC=C3C2=C1 SEHPSBSPFWAVPK-NRFANRHFSA-N 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 5
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 5
- 229940124639 Selective inhibitor Drugs 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000002055 immunohistochemical effect Effects 0.000 description 5
- 208000014674 injury Diseases 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 230000004807 localization Effects 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000007423 screening assay Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 238000002627 tracheal intubation Methods 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- 206010006458 Bronchitis chronic Diseases 0.000 description 4
- 206010011224 Cough Diseases 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 238000000636 Northern blotting Methods 0.000 description 4
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 4
- 108020004518 RNA Probes Proteins 0.000 description 4
- 239000003391 RNA probe Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000000556 agonist Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 201000009267 bronchiectasis Diseases 0.000 description 4
- 206010006451 bronchitis Diseases 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 208000007451 chronic bronchitis Diseases 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 231100000517 death Toxicity 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000012744 immunostaining Methods 0.000 description 4
- 238000000099 in vitro assay Methods 0.000 description 4
- 210000004969 inflammatory cell Anatomy 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000033001 locomotion Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 238000001543 one-way ANOVA Methods 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 210000002345 respiratory system Anatomy 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000001839 systemic circulation Effects 0.000 description 4
- 230000036962 time dependent Effects 0.000 description 4
- GFNNBHLJANVSQV-UHFFFAOYSA-N tyrphostin AG 1478 Chemical compound C=12C=C(OC)C(OC)=CC2=NC=NC=1NC1=CC=CC(Cl)=C1 GFNNBHLJANVSQV-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 3
- 108020005544 Antisense RNA Proteins 0.000 description 3
- 108090000994 Catalytic RNA Proteins 0.000 description 3
- 102000053642 Catalytic RNA Human genes 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 210000001552 airway epithelial cell Anatomy 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 3
- 239000002975 chemoattractant Substances 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 210000004081 cilia Anatomy 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000009786 epithelial differentiation Effects 0.000 description 3
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 3
- 239000003172 expectorant agent Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000005462 in vivo assay Methods 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229960001620 methohexital sodium Drugs 0.000 description 3
- 239000003595 mist Substances 0.000 description 3
- 230000002297 mitogenic effect Effects 0.000 description 3
- 230000000420 mucociliary effect Effects 0.000 description 3
- 239000006199 nebulizer Substances 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 238000009613 pulmonary function test Methods 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 230000000391 smoking effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 208000000884 Airway Obstruction Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 2
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 2
- 101500027527 Homo sapiens Transforming growth factor alpha Proteins 0.000 description 2
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 2
- 108010008211 N-Formylmethionine Leucyl-Phenylalanine Proteins 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 208000030880 Nose disease Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229940120646 Platelet-derived growth factor receptor kinase inhibitor Drugs 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 108091081021 Sense strand Proteins 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 238000005299 abrasion Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 208000037883 airway inflammation Diseases 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000035578 autophosphorylation Effects 0.000 description 2
- 238000000376 autoradiography Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 229940028978 brevital Drugs 0.000 description 2
- 230000005907 cancer growth Effects 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003112 degranulating effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000003419 expectorant effect Effects 0.000 description 2
- 229940066493 expectorants Drugs 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 102000056037 human MUC5AC Human genes 0.000 description 2
- 102000057041 human TNF Human genes 0.000 description 2
- 229940116978 human epidermal growth factor Drugs 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000003365 immunocytochemistry Methods 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000012678 infectious agent Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 229960001614 levamisole Drugs 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000004199 lung function Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 210000003254 palate Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 229960001412 pentobarbital Drugs 0.000 description 2
- 229960002275 pentobarbital sodium Drugs 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 208000010568 pulmonary mucoepidermoid carcinoma Diseases 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000019254 respiratory burst Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 210000004878 submucosal gland Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- ZIUSSTSXXLLKKK-KOBPDPAPSA-N (1e,4z,6e)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one Chemical compound C1=C(O)C(OC)=CC(\C=C\C(\O)=C\C(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 ZIUSSTSXXLLKKK-KOBPDPAPSA-N 0.000 description 1
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 1
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- WRGQSWVCFNIUNZ-GDCKJWNLSA-N 1-oleoyl-sn-glycerol 3-phosphate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP(O)(O)=O WRGQSWVCFNIUNZ-GDCKJWNLSA-N 0.000 description 1
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JIZRGGUCOQKGQD-UHFFFAOYSA-N 2-nitrothiophene Chemical group [O-][N+](=O)C1=CC=CS1 JIZRGGUCOQKGQD-UHFFFAOYSA-N 0.000 description 1
- CMLFRMDBDNHMRA-UHFFFAOYSA-N 2h-1,2-benzoxazine Chemical compound C1=CC=C2C=CNOC2=C1 CMLFRMDBDNHMRA-UHFFFAOYSA-N 0.000 description 1
- PGIFNMLEHONLJH-UHFFFAOYSA-N 4-[2-(2,7-dichloro-9h-fluoren-9-yl)ethoxycarbonylamino]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1NC(=O)OCCC1C2=CC(Cl)=CC=C2C2=CC=C(Cl)C=C21 PGIFNMLEHONLJH-UHFFFAOYSA-N 0.000 description 1
- FQNCLVJEQCJWSU-UHFFFAOYSA-N 6,7-dimethyl-2-phenylquinoxaline Chemical compound N1=C2C=C(C)C(C)=CC2=NC=C1C1=CC=CC=C1 FQNCLVJEQCJWSU-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- RTAPDZBZLSXHQQ-UHFFFAOYSA-N 8-methyl-3,7-dihydropurine-2,6-dione Chemical class N1C(=O)NC(=O)C2=C1N=C(C)N2 RTAPDZBZLSXHQQ-UHFFFAOYSA-N 0.000 description 1
- 108091005508 Acid proteases Proteins 0.000 description 1
- 208000036065 Airway Remodeling Diseases 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101800001382 Betacellulin Proteins 0.000 description 1
- 102400001242 Betacellulin Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229940121981 Carboxypeptidase inhibitor Drugs 0.000 description 1
- 101710127041 Carboxypeptidase inhibitor Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 108010062745 Chloride Channels Proteins 0.000 description 1
- 102000011045 Chloride Channels Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 206010014418 Electrolyte imbalance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- YCAGGFXSFQFVQL-UHFFFAOYSA-N Endothion Chemical compound COC1=COC(CSP(=O)(OC)OC)=CC1=O YCAGGFXSFQFVQL-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010051841 Exposure to allergen Diseases 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 229940123457 Free radical scavenger Drugs 0.000 description 1
- IECPWNUMDGFDKC-UHFFFAOYSA-N Fusicsaeure Natural products C12C(O)CC3C(=C(CCC=C(C)C)C(O)=O)C(OC(C)=O)CC3(C)C1(C)CCC1C2(C)CCC(O)C1C IECPWNUMDGFDKC-UHFFFAOYSA-N 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229940122853 Growth hormone antagonist Drugs 0.000 description 1
- HSRJKNPTNIJEKV-UHFFFAOYSA-N Guaifenesin Chemical compound COC1=CC=CC=C1OCC(O)CO HSRJKNPTNIJEKV-UHFFFAOYSA-N 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 208000003892 Kartagener syndrome Diseases 0.000 description 1
- 108010077861 Kininogens Proteins 0.000 description 1
- 102000010631 Kininogens Human genes 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 108010028275 Leukocyte Elastase Proteins 0.000 description 1
- 102000016799 Leukocyte elastase Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 208000032376 Lung infection Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 101710140999 Metallocarboxypeptidase inhibitor Proteins 0.000 description 1
- 102100022494 Mucin-5B Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000026344 Nasal disease Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 108010055723 PDGF receptor tyrosine kinase Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010003541 Platelet Activating Factor Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 101000762949 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) Exotoxin A Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000347485 Silurus glanis Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000000150 Sympathomimetic Substances 0.000 description 1
- 101150109894 TGFA gene Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000001800 adrenalinergic effect Effects 0.000 description 1
- 238000012387 aerosolization Methods 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000009285 allergic inflammation Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000002787 antisense oligonuctleotide Substances 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 208000025341 autosomal recessive disease Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000007630 basic procedure Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229940124630 bronchodilator Drugs 0.000 description 1
- 239000000168 bronchodilator agent Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- HFNQLYDPNAZRCH-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O.OC(O)=O HFNQLYDPNAZRCH-UHFFFAOYSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000006041 cell recruitment Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 208000013116 chronic cough Diseases 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 210000000215 ciliated epithelial cell Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- VVOLVFOSOPJKED-UHFFFAOYSA-N copper phthalocyanine Chemical compound [Cu].N=1C2=NC(C3=CC=CC=C33)=NC3=NC(C3=CC=CC=C33)=NC3=NC(C3=CC=CC=C33)=NC3=NC=1C1=CC=CC=C12 VVOLVFOSOPJKED-UHFFFAOYSA-N 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 238000011461 current therapy Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000004395 cytoplasmic granule Anatomy 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 239000007933 dermal patch Substances 0.000 description 1
- 229960002086 dextran Drugs 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000002337 electrophoretic mobility shift assay Methods 0.000 description 1
- 238000004836 empirical method Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- IECPWNUMDGFDKC-MZJAQBGESA-M fusidate Chemical compound O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C([O-])=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C IECPWNUMDGFDKC-MZJAQBGESA-M 0.000 description 1
- 229960004675 fusidic acid Drugs 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 210000003976 gap junction Anatomy 0.000 description 1
- 238000004868 gas analysis Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004704 glottis Anatomy 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 229960002146 guaifenesin Drugs 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000000076 hypertonic saline solution Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000003960 inflammatory cascade Effects 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 229940125369 inhaled corticosteroids Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 210000001847 jaw Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229960002683 methohexital Drugs 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000007491 morphometric analysis Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000000510 mucolytic effect Effects 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 210000003550 mucous cell Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 210000000492 nasalseptum Anatomy 0.000 description 1
- 229940105631 nembutal Drugs 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 238000012141 orotracheal intubation Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008723 osmotic stress Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000009527 percussion Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 235000020030 perry Nutrition 0.000 description 1
- 239000008249 pharmaceutical aerosol Substances 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical group 0.000 description 1
- 238000000554 physical therapy Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 201000009266 primary ciliary dyskinesia Diseases 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000003909 protein kinase inhibitor Substances 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 230000009325 pulmonary function Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- JWVCLYRUEFBMGU-UHFFFAOYSA-N quinazoline Chemical compound N1=CN=CC2=CC=CC=C21 JWVCLYRUEFBMGU-UHFFFAOYSA-N 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 229940124617 receptor tyrosine kinase inhibitor Drugs 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 210000001533 respiratory mucosa Anatomy 0.000 description 1
- 210000003019 respiratory muscle Anatomy 0.000 description 1
- 238000002644 respiratory therapy Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 210000005245 right atrium Anatomy 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000003728 serous cell Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 150000003440 styrenes Chemical class 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 229940032362 superoxide dismutase Drugs 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000001975 sympathomimetic effect Effects 0.000 description 1
- 229940064707 sympathomimetics Drugs 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000005092 tracheal tissue Anatomy 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- UOHFCPXBKJPCAD-UHFFFAOYSA-N tyrphostin 1 Chemical compound COC1=CC=C(C=C(C#N)C#N)C=C1 UOHFCPXBKJPCAD-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/02—Nasal agents, e.g. decongestants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/08—Bronchodilators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/10—Expectorants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/12—Mucolytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
Definitions
- This invention relates generally to the field of pulmonary treatment More particularly, the invention relates to inhibiting hypersecretion of mucus in lungs and airways by the administration of an EGF-R antagonist In addition, this invention also relates to methods for the development or assessment of candidate agents capable of inhibiting hypersecretion of mucus in the lungs
- the mucociliary system serves as the primary defense mechanism to move inhaled particles or infectious agents out of the airways in the lungs
- substances present in airway fluids serve to limit the toxicity of the particles and to inactivate infective agents
- the physical mechanism of coughing serves to expel the mucus from the airway passages (see e g , "Foundations of Respiratory Care,” Pierson and Kacmare , eds (1992) Churchill Livingstone Inc New York, New York, “Harrison's Principles of Internal Medicine", Fauci et al , eds (1997) 14th Edition, McGraw Hill, New York, New York)
- the mucociliary system consists of ciliated epithelial cells, epithelial goblet cells, and serous and mucous cells located in submucosal glands
- the cilia are surrounded by an aqueous layer (penciliary fluid) secreted into the lumen of the airway passage by the active transport of chloride and the passive movement of water across the epithelium
- the cilia make contact with the mucus floating on this aqueous layer, and via a unidirectional propelling motion provide for movement of mucus toward the glottis (see Pierson and Kacmarek, supra and Fauci, ef al , supra)
- Mucus is produced by the epithelial goblet cells and submucosal gland cells and is secreted into the lumen of the airway after degranulation
- ulcerative colitis Hypersecretion of mucus is the major symptom in patients with chronic obstructive pulmonary disease (COPD) and defines the condition (/ e chronic cough and sputum production) This condition alone affects 14 million Americans and can cause progressive disability and death It has been estimated that asthma affects at least 4% of the U S population and accounts for at least 2000 deaths annually (Pierson and Kucmarek, supra) During an acute asthmatic event, the bronchial walls swell, mucus volume increases and bronchial smooth muscle contracts, resulting in airway narrowing. As a result of hypersecretion in acute asthma, extensive mucus plugging can be a major cause of morbidity and mortality
- cystic fibrosis is an autosomal recessive disease that causes the airway mucosal cell to become unresponsive to cychc-AMP-dependent protein kinase activation of the membrane chloride ion channels (Pierson and Kacmarek, supra and Fauci, ef al , supra)
- the subsequent electrolyte imbalance reduces the level of hydration of the airway mucus, thus resulting in highly viscous mucus in the lungs of an individual afflicted with cystic fibrosis
- Hypersecretion obstructs the air passages of individuals with cystic fibrosis, further compromising lung function
- Other disease involving hypersecretion include chronic obstructive lung disorder (COPD)
- Mechanical intubation is often necessary in order to provide assisted ventilation to patients with various pulmonary diseases
- a tube is introduced via the oropharanx and placed in the trachea
- a balloon is inflated around the tube in the lower trachea, which may abrade the epithelium and cause goblet cell metaplasia Wounding of epithelium leads to repair processes, which can result in abundant mucus secretion
- Such prolonged tracheal intubation in patients can lead to deleterious effects due to hypersecretion
- bronchodilators e g , methylxanthines, sympathomimetics with strong ⁇ 2 adrenergic stimulating properties, anticholmergics
- systemic or inhaled corticosteroids primarily in asthma, liquefaction of the mucus by oral administration of expectorants, e g guaifenesin, and aerosol delivery of "mucolytic" agents, e g water, hypertonic saline solution
- a newer therapy for cystic fibrosis is the administration of DNAse to target the DNA- ⁇ ch mucus or sputum (Shak, ef al (1990) Proc Natl Acad (USA) 87 9188-9192, Hubbard, R C et al (1991) N Engl J Med 326 812)
- a primary object of the invention is to provide a method of treating diseases involving hypersecretion of mucus in lungs
- Another object of the invention is to provide formulations useful in the treatment of diseases that result in hypersecretion of mucus
- Yet another object of the invention is to provide an in vitro assay for the screening of candidate agents that inhibit hypersecretion of mucus, where the method involves the steps of (i) contacting an in vitro model of goblet cell proliferation with EGF or the functional equivalent thereof, (n) subsequently contacting the in vitro model with a candidate agent, and (m) assessing goblet cell proliferation, wherein inhibition of goblet cell proliferation is indicative of the candidate agent's therapeutic potential
- Another object of the invention is to provide an in vivo assay for the screening of candidate agents that inhibit hypersecretion of mucus, where the method involves (i) creating an animal model of hypersecretory pulmonary disease by inducing EGF-R, e g with tumor necrosis factor-alpha (TNF- ⁇ ), (n) stimulating the induced EGF-R with its ligand, e g transforming growth factor alpha (TGF- ) or EGF, to produce mu n producing goblet cells, (in) treating with a candidate agent, and (iv) assessing goblet cell proliferation or mucus secretion, wherein an inhibition of goblet cell proliferation or mucus secretion is indicative of the candidate agent's therapeutic potential
- a further object of the invention is to provide in vitro and in vivo assays for the screening of EGF-R antagonists that inhibit hypersecretion of mucus
- An advantage of the invention is that it provides a means for preventing excessive formation of mucus in pulmonary airways
- a feature of the invention is that a range of different types of antagonists can be used to block the effects of EGF and/or TGF- ⁇ and their interaction with EGF-R
- An aspect of the invention is formulations of EGF antagonists for reducing formation of mucus secretion in the airways of a mammalian patient, preferably a human patient.
- Another object of the invention is a method of pulmonary delivery of EGF antagonists for reducing mucus secretions in the airways of a mammalian patient, preferably a human patient.
- Another object of the invention is to provide a method for treating a range of different diseases which have as a symptom the excess formulation of mucus secretions in the airways. These diseases include, without limitation, chronic bronchitis, acute asthma, cystic fibrosis, bronchiectasis, chronic obstructive lung disease, hypersecretion resulting from epithelial damage such as allergic stimuli or mechanical abrasions, and nasal hypersecretion.
- Figure 1A is a western blot of EGF-R in NCI-H292 and in A431 cells.
- Figure 1B immunocytochemical analysis with anti-EGF-R antibody in cultures of NCI-H292 cells.
- Figure 1C Northern analysis of EGF-R in NCI-H292 cells.
- Figure 2 Alcian blue/PAS staining of NCI-H292 cells for identification of mucin glycoproteins.
- FIG 4A and 4B Immunohistochemical analysis of EGF-R with an anti-EGF-R antibody in pathogen-free rats.
- Figure 4A TNF ⁇ -treated rats.
- Figure 4B ovalbumin-sensitized rats.
- Figure 5 is a graph depicting the effect of EGF-R tyrosine kinase inhibitor (BIBX1522) on production of goblet cells (expressed as % of stained area of airway epithelium occupied by Alcian blue/PAS-positive stained cells).
- compositions and methods are provided for the treatment of airway mucus hypersecretion by administering therapeutic amounts of EGF antagonists, preferably kinase inhibitors.
- EGF antagonists preferably kinase inhibitors.
- the antagonists may be in the form of small molecules, antibodies, or portions of antibodies that bind to either EGF or its receptor.
- airway hypersecretory diseases e.g. chronic bronchitis, bronchiectasis, cystic fibrosis, acute asthma, COPD, etc.
- mucin synthesis in airways is increased and mucus hypersecretion occurs.
- the secreted mucus results in airway obstruction, an effect that causes death in these diseases.
- an evolutionary sequence of goblet cell production may be based on the expression of EGF-R Stimulation with TNF ⁇ induces intense EGF-R staining of non-granulated secretory cells, their subsequent activation by EGF-R gands causes progressive staining for mucous glycoconjugates in the cytoplasm, and the cells become "pre-goblet” and then "goblet” cells
- EGF-R activation promotes selective cell differentiation, but not proliferation
- Goblet cells are apparently derived from non-granulated secretory cells that express EGF-R and are stimulated by EGF-R gands to produce mucins
- the EGF-R may be stimulated by other signaling mediators
- the EGF-R may be stimulated by other signaling mediators
- Proinflammatory cytokme-activated neutrophils and cigarette smoke are shown to cause mucin synthesis in human bronchial epithelial cells via ligand-independent activation of EGF-R, implicating recruited neutrophils and cigarette smoke as regulators of epithelial cell differentiation that result in abnormal induction of mucin-producing cells in airways
- Neutrophils activated by a variety of stimuli, including IL-8, N-formyl-methionyl-leucyl- phenylalanine, TNF- ⁇ , cigarette smoke or H 2 0 2 upregulate mucin expression in epithelial cells, which synthesis is inhibited by EGF-R inhibitors
- Neutrophils are also capable of producing the EGF-R gands, EGF and TGF ⁇
- epithelial cells including IL-8, N-formyl-me
- Epithelial damage is a common finding in studies of patients even with mild asthma, and the damage is increasingly related to worsening of clinical symptoms
- Epithelial damage produced by the allergic response induces EGF-R activation, which results in abnormal goblet cell production
- EGF-R is implicated in epithelial damage, for example the "airway remodeling" that occurs in asthma, repair and wound closure
- Mechanical epithelial damage and epithelial injury in asthma may involve a similar EGF-R cascade, resulting in abnormal growth of epithelial secretory cells
- Hypersecretion is also an important manifestation of inflammatory diseases of the nose
- nasal goblet cells are "challenged” by inducing goblet cell degranulation utilizing a neutrophil-dependent mechanism, expression of EGF-R and mucins are strongly upregulated These events were associated with regranulation of the goblet cells
- inflammation such as stimulation of neutrophil infiltration, causes goblet cell degranulation and mucin secretion, up-regulation and activation of EGF-R re-supphes the airway epithelium with mucins
- EGF epidermal growth factor
- epidermal growth factor or "EGF” is meant a protein or portion thereof having biological activity characterized by mitogenic activity on epithelial cells (e g , Cohen (1986) Biosciences Reports 6(12) 1017, Aaronson, S A , “Growth Factors and Cancer,” Science (1991 ) 254 1146-1153)
- epithelial cells e g , Cohen (1986) Biosciences Reports 6(12) 1017, Aaronson, S A , “Growth Factors and Cancer,” Science (1991 ) 254 1146-1153
- human epidermal growth factor for example as described by Urdea ef al (1983) Proc Nat Acad Sci 80 7461-7465
- proteins of portions thereof which are the functional equivalent of EGF in terms of the biological response elicited by EGF
- EGF-R epidermal growth factor receptor
- a protein a portion thereof capable of binding EGF protein or a portion thereof Exemplary is the human epidermal growth factor receptor (see Ullrich ef al (1984) Nature 309418-425, Genbank accession number NM_005228)
- the binding of the EGF hgand activates the EGF-R (e g resulting in activation of mtracellular mitogenic signaling, autophosphorylation of EGF-R)
- EGF-R epidermal growth factor receptor
- hgands include, but are not limited to, TGF- ⁇ , betacellulin, amphireguhn, hepann-binding EGF (HB-EGF) and neureguhn (also known as hergu n) (Strawn and Shawver (1998) Exp -Opm Invest Drugs 7(4)553-573,
- EGF-R antagonist any agent capable of directly or indirectly inhibiting the effect of EGF-R, particularly the effect of EGF-R on goblet cell proliferation or hypersecretion of mucus by goblet cells
- EGF-R can be activated through hgand-dependent and hgand-independent mechanisms, resulting in either autophosphorylation or trans-phosphorylation, respectively
- EGF-R antagonists of interest may inhibit either or both of these mechanisms
- binding of TNF- ⁇ to the EGF-R results in a ligand-dependent phosphorylation, which may be blocked by an antibody that binds EGF-R, thereby preventing the interaction of EGF with a hgand that would activate the EGF receptor
- Examples of such antibodies are described by Goldstein et al (1995) Clin Cancer Res 1 1311-1318, Lo ⁇ mer ef a/ (1995) Clin Cancer Res 1 859-864, Schmidt and Wels (1996) Br J Cancer 74 853-862 Small molecule tyrosine kinas
- An EGF-R antagonist may be an antibody that binds to a factor that stimulates EGF production or EGF-R production, thereby inhibiting promotion of goblet cell proliferation by EGF (/ e an inhibitor of the phosphorylation cascade that phosphorylates EGF-R)
- EGF an inhibitor of the phosphorylation cascade that phosphorylates EGF-R
- a fusion protein of TGFa- Pseudomonas exotoxin 40 is described by Arteaga ef al (1995) Cancer Res 54 4703-4709
- the EGF-R antagonist is an inhibitor of the tyrosine kinase activity of EGF-R, particularly small molecule inhibitors having selective action on EGF-R as compared to other tyrosine kinases - preferred small molecules block the natural EGF receptor in a mammal, preferably a human and have a molecular weight of less than 1 kD
- Inhibitors of EGF and EGF-R include, but are not limited to, tyrosine kinase inhibitors such as quinazohnes, such as PD 153035, 4-(3-chloroan ⁇ l ⁇ no) quinazoline, or CP-358,774, pyridopynmidmes, py ⁇ midopy ⁇ midines, pyrrolopy ⁇ midines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopy ⁇ midines (Shawn and Shawver, supra ), 4-(phenylam ⁇ no)-7H-pyrrolo[2,3-d] py ⁇ midines (Traxler ef al , (1996) J Med Chem 39 2285-2292), curcumm (diferuloyl methane) (Laxmin arayana, et al, (1995), Carcinogen 16 1741-1745), 4,5-b ⁇ s (4-fluoroan ⁇ hno)
- treatment means obtaining a desired pharmacologic and/or physiologic effect
- the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease
- Treatment covers any treatment of a disease in a mammal, particularly a human, and includes
- the invention is directed toward treating patients with pulmonary or airway disease and is particularly directed toward treating patients' hypersecretion of mucus, / e preventing, inhibiting or relieving hypersecretion of mucus
- the invention is directed toward decreasing mucus or sputum in the airways, inhibiting infection by pathological organisms, alleviating cough, and preventing hypoxia due to airway plugging
- treatment is intended to mean providing a therapeutically detectable and beneficial effect on a patient suffering from a pulmonary disease involving hypersecretion of mucus
- treatment shall mean preventing, alleviating, and/or inhibiting hypersecretion of mucus with a compound selected from the group consisting of EGF and/or EGF-R antagonists such as antibodies, protein tyrosine kinase inhibitors and antisense molecules and the like
- An alternative treatment may comprise prevention of EGF-R expression in airway, thereby blocking the pathway at an earlier stage
- reagents that block binding of TNF ⁇ to its receptor may prevent upregulation of EGF-R
- Treatment includes preventing or inhibiting infections by pathological agents caused by and/or related to hypersecretion of mucus
- antibody an immunoglobu n protein that is capable of binding an antigen
- Antibody as used herein is meant to include antibody fragments, e g F(ab')2, Fab', Fab, capable of binding the antigen or antigenic fragment of interest
- the binding of the antibody to the antigen inhibits the activity of EGF or EGF-R
- humanized antibody is used herein to describe complete antibody molecules, / e composed of two complete light chains and two complete heavy chains, as well as antibodies consisting only of antibody fragments, e g Fab, Fab' , F (ab') 2, and Fv, wherein the CDRs are derived from a non-human source and the remaining portion of the Ig molecule or fragment thereof is derived from a human antibody, preferably produced from a nucleic acid sequence encoding a human antibody
- human antibody and “humanized antibody” are used herein to describe an antibody of which all portions of the antibody molecule are derived from a nucleic acid sequence encoding a human antibody Such human antibodies are most desirable for use in antibody therapies, as such antibodies would elicit little or no immune response in the human patient
- chime ⁇ c antibody is used herein to describe an antibody molecule as well as antibody fragments, as described above in the definition of the term “humanized antibody "
- the term “chime ⁇ c antibody” encompasses humanized antibodies Chime ⁇ c antibodies have at least one portion of a heavy or light chain ammo acid sequence derived from a first mammalian species and another portion of the heavy or light chain ammo acid sequence derived from a second, different mammalian species
- the variable region is derived from a non-human mammalian species and the constant region is derived from a human species
- the chime ⁇ c antibody is preferably produced from a nucleotide sequence from a non-human mammal encoding a variable region and a nucleotide sequence from a human encoding a constant region of an antibody
- binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide Antibody binding to its epitope on a specific polypeptide is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest
- Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level, e g 10% or less of the binding shown to the polypeptide of interest Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e g by use of appropriate controls
- detectable labeled antibody detectably labeled anti-EGF
- detectably labeled anti-EGF fragment is meant an antibody (or antibody fragment that retains binding specificity), having an attached detectable label The detectable label is normally attached by chemical conjugation, but where the label is a polypeptide
- Detectable labels may be selected from a variety of such labels known in the art, but normally are radioisotopes, fluorophores, enzymes, e g horseradish peroxidase, or other moieties or compounds that either emit a detectable signal (e g radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate
- Various detectable label/substrate pairs e g horseradish peroxidase/diaminobenzidme, avidin/streptavidm, luciferase/lucifenn
- methods for labeling antibodies and methods for using labeled antibodies to detect an antigen are well known in the art (for example, see Harlow and Lane, eds (Antibodies A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) THERAPEUTIC METHODS
- the present invention provides a method of treating pulmonary hypersecretion by administering therapeutic amounts of EGF-R antagonists
- Any disease and particularly and pulmonary disease characterized by hypersecretion of mucus or accumulation of pathological levels of mucus may be treated by the methods described herein
- pulmonary hypersecretory diseases that may be treated by this method include, but are not limited to, chronic obstructive lung diseases, such as chronic bronchitis, inflammatory diseases such as asthma, bronchiectasis, pulmonary fibrosis, COPD, diseases of nasal hypersecretion, e g nasal allergies, and other hypersecretory diseases
- Genetic diseases such as cystic fibrosis, Kartagener syndrome, alpha-1-ant ⁇ trypsm deficiency, familial non-cystic fibrosis mucus spissation of respiratory tract, are intended to be included as well
- Antagonists that directly target EGF or EGF-R are preferred
- any factor or cell involved in the biological cascade that results in EGF-R promoting goblet cell proliferation may be targeted for inhibition, e g TGF- ⁇ antagonists
- a cascade begins during an inflammatory response when cells such as mast cells or neutrophils release TNF- ⁇ , which then promotes EGF-R expression Stimulation of EGF-R, e g by its ligand EGF, in turn triggers goblet cell proliferation
- any cells or factors involved in the cascade, such as in the TNF- ⁇ pathway may be targeted for antagonist activity
- the EGF-R antagonist administered in the therapeutic method may be in any form
- the EGF-R antagonist may be in the form of a small molecule (/ e , antisense ohgonucleotide, tyrosine kinase inhibitor, efc ), antibodies or portion of antibodies that bind to EGF, TGF ⁇ or EGF-R
- SMALL MOLECULE EGF-R ANTAGONISTS Tyrosine kinase inhibitors that act on the EGF receptor, and that are selective for the EGF-R, are known in the art, and may be used in the subject methods Examples are described above, and of such may include BIBX1522 (Boeh ⁇ nger Ingelheim, Inc , Ingelheim, Germany), CGP59326B (Novartis Corporation, Basel, Switzerland), 4-am ⁇ noqu ⁇ nazohne EGF-R inhibitors (described in U S Patent no 5,760,041), substituted styrene compounds which can also be a naphthalene, an mdane or a benzoxazine, including nitnle and molononit ⁇ le compounds (described in U S Patent no 5,217,999), the inhibitors disclosed in U S Patent no 5,773,476, potato carboxypeptidase inhibitor (PCI), a 39-am ⁇ no acid protease inhibitor with three disul
- Preferred tyrosine kinase inhibitors are selective for EGF receptor, / e the EGF-R is inhibited to a greater degree than other cell surface receptors having tyrosine kinase activity Selectivity is enhanced by the methods of formulation and drug delivery, e g where the inhibitor is preferentially delivered to inflamed airways, etc
- Typical dosages for systemic administration range from 0 1 ⁇ g to 100 milligrams per kg weight of subject per administration Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects Some of the specific compounds are more potent than others Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means A preferred means is to measure the physiological potency of a given compound, for example with the in vitro and in vivo tests described herein
- Antibodies as EGF-R antagonists are of particular interest (e g Vilona, ef al , American Journal of Pathology 151 1523)
- Antibodies to EGF or EGF-R are produced by immunizing a xenogeneic immunocompetent mammalian host, including murine, rodentia, lagomorpha, ovine, porcine, bovine, efc with EGF or EGF-R or portions thereof
- a xenogeneic immunocompetent mammalian host including murine, rodentia, lagomorpha, ovine, porcine, bovine, efc with EGF or EGF-R or portions thereof
- human EGF or EGF-R or portions thereof are used as the immunogen
- the choice of a particular host is primarily one of convenience
- Immunizations are performed in accordance with conventional techniques, where the immunogen may be injected subcutaneously, intramuscularly, intrapentoneally, travascularly, efc into the host animal
- EGF or EGF-R intrapentoneally every other day will be used as an immunogen
- the injections may be with or without adjuvant, e g complete or incomplete Freund's adjuvant, specol, alum, efc
- the antiserum may be harvested in accordance with conventional ways to provide polyclonal antisera specific for EGF or the EGF-R
- Either monoclonal or polyclonal antibodies, preferably monoclonal antibodies, are produced from the immunized animal
- Polyclonal antisera may be harvested from serum by conventional methods from the animals after completion of the immunization schedule
- lymphocytes are harvested from the appropriate lymphoid tissue, e g spleen, draining lymph node, efc , and fused with an appropriate fusion partner, usually a myeloma line, producing a hybndoma secreting a specific monoclonal antibody
- Screening clones of hybndomas for the antigenic specificity of interest is performed in accordance with conventional methods
- EGF so as to inhibit binding of EGF to EGF-R, e g an antibody that specifically binds to the extracellular domain of EGF-R thereby preventing binding of EGF
- Such antibodies may be made by conventional methodology described above, or are commercially available.
- Examples of antibodies that would function as an EGF antagonist include, but are not limited to, the neutralizing anti-EGF-R monoclonal antibody C225 (Kawamoto ef at (1983) Proc. Natl Acad. Sci. (USA) 80:1337-1341 ; Petit et al. (1997) J. Path. 151:1523-153, produced by ImClone Systems New York, NY) and the anti-EGF-R monoclonal antibody EMD55900 (also called Mab 425), (Merck, Darmstadt, Germany).
- the subject antibodies may be produced as a single chain, instead of the normal multimeric structure.
- Single chain antibodies are described in Jost ef al. (1994) J.B.C. 269:26267-73, and others.
- DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine.
- the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
- the humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example, International Patent Applications WO 90/10077 and WO 90/04036).
- the antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1 , CH2, CH3, hinge domains, and/or the framework residues with the corresponding human sequence (see WO 92/02190).
- Ig cDNA for construction of chimeric immunoglobulin genes is also known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521 ).
- mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
- the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Patent nos. 4,683,195 and 4,683,202).
- a library is made and screened to isolate the sequence of interest.
- the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
- human constant regions genes may be found in Kabat ef al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The chimeric, humanized antibody is then expressed by conventional methods.
- Antibody fragments such as Fv, F(ab') 2 and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage.
- a truncated gene is designed.
- a chimeric gene encoding a portion of the F(ab') 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
- An individual having a hypersecretory mucus disease may initially be administered amounts of EGF-R antagonist in the range of about 20 milligrams (mg) to about 400 mg per kilogram weight of patient twice daily, e.g. by inhalation.
- EGF-R antagonist in the range of about 20 milligrams (mg) to about 400 mg per kilogram weight of patient twice daily, e.g. by inhalation.
- the subject therapeutic agents are antisense molecules specific for human sequences coding for EGF or EGF-R
- the administered therapeutic agent may be antisense ohgonucleotides, particularly synthetic ohgonucleotides having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA
- the antisense sequence is complementary to the mRNA of the targeted EGF or EGF-R genes, and inhibits expression of the targeted gene products (see e g Nyce ef al (1997) Nature 385 720)
- Antisense molecules inhibit gene expression by reducing the amount of mRNA available for translation, through activation of RNAse H or ste ⁇ c hindrance
- One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences from a single targeted gene, or sequences that complement several different genes
- a preferred target gene is EGF-R or EGF
- the gene sequence may be accessed through public databases (human epidermal growth factor, Genbank accession no K01166, human mRNA for precursor of epidermal growth factor receptor, Genbank accession no X00588) Generally, the antisense sequence will have the same species of origin as the animal host
- Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the vector is introduced and expressed in the targeted cells
- the transcnptional initiation will be oriented such that the antisense strand is produced as an RNA molecule
- the anti-sense RNA hybridizes with the endogenous sense strand mRNA, thereby blocking expression of the targeted gene
- the native transcnptional initiation region, or an exogenous transcnptional initiation region may be employed
- the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome
- Many strong promoters that are active in muscle cells are known in the art, including the ⁇ -actm promoter, SV40 early and late promoters, human cytomegaiovirus promoter, retroviral LTRs, efc Transcription vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucle
- the antisense molecule is a synthetic o gonucleotide
- Antisense ohgonucleotides will generally be from about 7 to 500, usually from about 12 to 50 nucleotides, more usually from about 20 to 35 nucleotides, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like It has been found that short ohgonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner ef al (1996) Nature Biotechnology 14 840-844)
- a specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence It has been shown that the 5' region of mRNA is particularly susceptible to antisense inhibition
- Selection of a specific sequence for the ohgonucleotide may use an empirical method, where several candidate sequences are assa
- Antisense ohgonucleotides may be chemically synthesized by methods known in the art (see Wagner ef al (1993) supra and Mil gan ef a/ , supra ) Preferred ohgonucleotides are chemically modified from the native phosphodiester structure, in order to increase their mtracellular stability and binding affinity A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases
- Ohgonucleotides may additionally comprise a targeting moiety that enhances uptake of the molecule by cells
- the targeting moiety is a specific binding molecules, e g an antibody or fragment thereof that recognizes molecules present on the surface of lung epithelial cells, particularly epithelial cells containing EGF-R
- Bispecific antibodies, chimeric antibodies and single chain antibodies are known in the art
- non-human antibodies can be humanized in various ways
- Linkage between the ohgonucleotide and targeting moiety may use any conventional method, for example by disulfide, amide or thioether bonds, depending on the chemistry of the ohgonucleotide backbone
- the linkage will be cleaved inside the cell to liberate the ohgonucleotide
- Ohgonucleotides can be conjugated to hydrophobic residues, e g cholesterol, to protect from nucleases and to improve transport across cell membranes Alternatively, conjugation to poly-L-lysine or other polyammes may also enhance delivery to the cell A further modification that can be made is the addition of an intercalating component, such as acndine, capable of intercalating into the target mRNA and stabilizing the resultant hybrid Antisense ohgonucleotides may be transfected in combination with an enzyme(s) that will degrade antisense-mRNA complexes in the cell, e g RNase-H Any protein or enzyme that can preferentially degrade or sequester the antisense-mRNA duplex may be similarly useful
- catalytic nucleic acid compounds e g ribozymes, anti-sense conjugates, etc may be used to inhibit gene expression
- Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al (1995) Nucl Acids Res 234434-42)
- Examples of ohgonucleotides with catalytic activity are described in WO 9506764
- Conjugates of anti-sense ohgonucleotides with a metal complex, e g terpy ⁇ dylCu(ll) capable of mediating mRNA hydrolysis are described in Bashkm ef al (1995) Appl Biochem Biotechnol 54 43-56
- EGF-R antagonists may be provided in solution or in any other pharmacologically suitable form for administration, such as a hposome suspension
- the appropriate antibodies or other form of anti- EGF are formulated for administration in a manner customary for administration of such materials Typical formulations are those provided in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA
- the route of administration will be selected based on the compound being administered, the status of the patient and disease that is being treated Where there is hypersecretion of mucus, a compound may be administered through different routes depending on the severity of the disease, e g emergency situations may require i v administration, acute but not life threatening situation may be treated orally, while chronic treatment can be administered by aerosol
- the EGF antagonist may be administered by injection, including intramuscular, intravenous (IV), subcutaneous or peritoneal injection, most preferably IV and local injections
- IV intravenous
- other modes of administration may also be used provided means are available to permit the EGF-R antagonist to enter the systemic circulation, such as transmucosal or transdermal formulations, which can be applied as suppositories, skin patches, or mtranasally
- Any suitable formulation that effects the transfer of the EGF-R antagonist to the bloodstream or locally to the lungs may properly be used
- suitable formulations generally comprise aqueous solutions or suspensions using physiological saline, Hank's solution, or other buffers optionally including stabilizing agents or other minor components
- suitable formulations can also be used
- the EGF-R antagonist may also be supplied in lyophilized form and reconstituted for administration
- Transmucosal and transdermal administrations generally include agents that facilitate passage through the mucosal or dermal barrier, such as bile, salts, fusidic acid and its analogs, various detergents and the like
- suitable enteric coatings are formulated to permit the EGF-R antagonist to survive the digestive tract
- the nature of the formulation will depend to some extent on the nature of the EGF-R antagonist chosen
- a suitable formulation is prepared using known techniques and principles of formulation well known to those skilled in the art
- the percentage of EGF-R antagonists contained in a particular pharmaceutical composition will also depend on the nature of the formulation, the percentage of an EGF-R antagonist that is an antibody will typically vary over a wide range from about 1 % by weight to about 85% by weight
- Useful delivery systems include Sendai virus-liposome delivery systems (Rapaport and Shai
- liposomes as a delivery vehicle is one method of interest for use with EGF-R antagonists.
- the liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly.
- the liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like.
- Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc.
- the lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine.
- the remaining lipid will normally be neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
- neutral lipids such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
- the procedure described by Kato et al. (1991) J. Biol. Chem. 266:3361 may be used.
- the EGF-R antagonist is encapsulated in a sterically stabilized
- stealth liposomes e.g. pegylated liposomes.
- liposomes When such liposomes are injected i.v., they remain in the circulation for long periods.
- Postcapillary venular gap junctions open during airway inflammation and allow fluid accumulation and permit molecules, e.g. complement, kininogen, to enter tissues, initiating inflammatory cascades.
- Such inflammation allows liposomes and their contents to be deposited selectively in the inflamed tissue (Zhang ef al. (1998) Pharm Res 15:455-460).
- EGF-R antagonists may be administered to the afflicted patient by means of a pharmaceutical delivery system for the inhalation route.
- the compounds may be formulated in a form suitable for administration by inhalation.
- the pharmaceutical delivery system is one that is suitable for respiratory therapy by topical administration of EGF-R antagonists thereof to mucosal linings of the bronchi.
- This invention can utilize a system that depends on the power of a compressed gas to expel the EGF-R antagonists from a container. An aerosol or pressurized package can be employed for this purpose.
- aerosol is used in its conventional sense as referring to very fine liquid or solid particles carries by a propellant gas under pressure to a site of therapeutic application.
- the aerosol contains the therapeutically active compound, which can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and a propellant.
- the aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols employed in the present invention are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a patient.
- propellants include, but is not limited to, hydrocarbons or other suitable gas.
- the dosage unit may be determined by providing a value to deliver a metered amount.
- the present invention can also be carried out with a nebulizer, which is an instrument that generates very fine liquid particles of substantially uniform size in a gas
- a nebulizer which is an instrument that generates very fine liquid particles of substantially uniform size in a gas
- a liquid containing the EGF-R antagonists is dispersed as droplets
- the small droplets can be carried by a current of air through an outlet tube of the nebulizer The resulting mist penetrates into the respiratory tract of the patient
- a powder composition containing EGF-R antagonists or analogs thereof, with or without a lubricant, carrier, or propellant, can be administered to a mammal in need of therapy
- This embodiment of the invention can be carried out with a conventional device for administering a powder pharmaceutical composition by inhalation
- a powder mixture of the compound and a suitable powder base such as lactose or starch may be presented in unit dosage form in for example capsular or cartridges, e g gelatin, or blister packs, from which the powder may be administered with the aid of an inhaler
- Combination therapies may be used to treat hypersecretory pulmonary disease
- EGF-R antagonists may be combined with conventional treatment for alleviation of hypersecretion, such as bronchiodilators, corticosteroids, expectorants, mucolytic agents and the like to facilitate mucociliary clearance
- a formulation of the present invention by injection (e g , intravenous) or by inhalation Patients which have large amounts of mucus in the lungs cannot, in general, be treated initially by inhalation This is due to the fact that the patient's lungs are sufficiently obstructed that inhaling aerosolized formulation into the lungs may not be particularly effective
- administration by inhalation is preferred
- Administration by inhalation is preferred because smaller doses can be delivered locally to the specific cells which are most in need of treatment By delivering smaller doses, any adverse side effects are eliminated or substantially reduced By delivering directly to the cells which are most in need of treatment, the effect of the treatment will be realized more quickly
- Antagonists of the present invention can be formulated in basically three different types of formulations for inhalation
- antagonists of the invention can be formulated with low boiling point propellants
- Such formulations are generally administered by conventional meter dose inhalers (MDI's)
- MDI's can be modified so as to increase the ability to obtain repeatable dosing by utilizing technology which measures the spiratory volume and flow rate of the patient as discussed within U S Patents 5,404,871 and 5,542,410
- the agonists of the present invention can be formulated in aqueous or ethanohc solutions and delivered by conventional nebulizers
- solution formulations are aerosolized using devices and systems such as disclosed within U S Patent 5,497,763, 5,544,646, 5,718,222, and 5,660,166
- agonist compounds of the present invention can be formulated into dry powder formulations Such formulations can be administered by simply inhaling the dry powder formulation after creating an aerosol mist of the powder Technology for carrying such out is described within U S Patent 5,775,320 issued July 7, 1998 and U S Patent 5,740,794 issued April 21 , 1998
- U S Patent 5,775,320 issued July 7, 1998 U S Patent 5,740,794 issued April 21 , 1998
- applicants point out that these patents cite other publications in intrapulmonary drug delivery and such publications can be referred to for specific methodology, devices and formulations which could be used in connection with the delivery of agonists of the present invention
- each of the patents are incorporated herein by reference in their entirety for purposes of disc
- Screening assays may be used to identify bioactive candidate agents that are EGF antagonists Of particular interest are screening assays for agents that have a low toxicity for human cells
- assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like
- the purified EGF or EGF-R protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transporter function, etc
- agent as used herein describes any molecule, e g protein or pharmaceutical, with the capability of altering or inhibiting the physiological function of EGF or EGF-R
- a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, / e at zero concentration or below the level of detection
- Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds
- Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized ohgonucleotides and ohgopeptides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, este ⁇ fication, amidification, efc to produce structural analogs
- the screening assay is a binding assay
- the label can directly or indirectly provide a detectable signal
- Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e g magnetic particles, and the like
- Specific binding molecules include pairs, such as biotin and streptavidm, digoxin and antidigoxin, etc
- the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures
- reagents may be included in the screening assay These include reagents like salts, neutral proteins, e g albumin, detergents, etc that are used to facilitate optimal protem- protein binding and/or reduce non-specific or background interactions Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc may be used The mixture of components are added in any order that provides for the requisite binding
- Incubations are performed at any suitable temperature, typically between 4 and 40° C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening Typically between 0 1 and 1 hours will be sufficient
- the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of hypersecretory disease in formulations described herein Depending upon the manner of introduction, the compounds may be formulated in a variety of ways described herein
- the concentration of therapeutically active compound in the formulation may vary from about 0 1-100% by weight
- the appropriate dosage level will also vary depending on a number of factors including the nature of the subject to be treated, the particular nature of the hypersecretory condition to be treated and its severity, the nature of the EFGR antagonist used as active ingredient, the mode of administration, the formulation, and the judgement of the practitioner For example, when antibodies are administered by themselves such as anti-EGF or EGF-R in an mjectable formulation, the dosages will be in the range of 20 mg/kg to about 40 mg/kg at a single dosage Repeated administration over a period of days may be required or administration by intravenous means may be continuous For chronic conditions, administration may be continued for longer periods as necessary
- Efficacy of the dosing regime will be determined by assessing for improved lung function in the patient This assessment may include viscoelasticity measurements of sputum, improvements in pulmonary function, including improvements in forced exploratory volume of sputum and maximal midexpiratory flow rate
- the aforementioned therapeutic regime can be given in conjunction with adjunct therapies such as antibiotics, DNAse I or other current therapies for the treatment of hypersecretory pulmonary disease If antibiotics are co-administered as part of the patient's therapy, bacterial quantitation following therapy can be included to assess the efficacy of the treatment by decreased bacterial growth, indicating decreased viscosity of mucus or sputum and increase of the mucus or sputum lung clearance
- Pulmonary function tests as well as diagnostic tests for the clinical progression of pulmonary hypersecretory disease, are known to those individuals with skill in this art
- Standard pulmonary function tests include airway resistance (AR), forced vital capacity (FVC), forced expiratory volume in 1 second (FEV(1)), forced midexpiratory flow, and peak expiratory flow rate (PEFR)
- Other pulmonary function tests include blood gas analysis, responses to medication, challenge and exercise testing, measurements of respiratory muscle strength, fibro-optic airway examination, and the like
- Some basic procedures for studying the properties of mucus include rheology, e g with the use of a magnetic microrheometer, adhesivity to characterize the forces of attraction between an adherent surface and an adhesive system by measuring the contact angle between a mucus drop and a surface Mucus transport by cilia can be studied using conventional techniques, as well as direct measurement, / e in situ mucus clearance Transepithehal potential difference, the net result of the activity of the ion-transport system of the pulmonary epithel
- the patient to be treated can be a primate, such as a human, or any other animal exhibiting the described symptoms While the method of the invention is especially adapted for the treatment of a human patient, it will be understood that the invention is also applicable to veterinary practice
- in vitro assays are used to assess the therapeutic potential of candidate agents to inhibit goblet cell proliferation, / e whether such agents are active as an EGF antagonist
- such assays will comprise the following steps (i) contacting an in vitro model of goblet cell proliferation with EGF or the functional equivalent thereof, (n) subsequently contacting the in vitro model with a candidate agent, and (in) assessing goblet cell proliferation, wherein an inhibition of goblet cell proliferation is indicative of the candidate agent's therapeutic potential
- rat tracheal cells can be isolated and maintained in culture as described in Guzman et al (1995) 217 412-419 Briefly, the rat tracheal cells are plated onto collagen gel coated semipermeable membranes, initially cultured submerged in media, and subsequently maintained with an air/hquid interface
- in vitro cells include primary human bronchial cells (available from Clonetics, San Diego), NCI-H292 cells (ATCC CRL-1848), and A431 cells (ATCC CRL-1555)
- the in vitro culture is contacted with EGF and with a candidate agent
- the candidate agent may be contacted with the culture prior to, concurrently with, or subsequently to the addition of EGF depending on the endpoint to be assessed and the nature of the candidate agent
- the cultured cells are assessed for inhibition of goblet cell proliferation relative to controls
- molecular or biochemical markers may be used to assess goblet cell proliferation
- molecular or biochemical markers include, but are not limited to, gene expression or protein expression characteristic of goblet cells
- Certain mucin genes e g MUC5B (Desseyn et al (1997) J Biol Chem 272 3168-3178) are expressed in the airway, and have a gene product highly represented in mucus Expression of mucin genes provides a suitable marker for determining production of mucus Mucin gene expression may be assessed by conventional technology such as northern blot analysis, polymerase chain reaction (PCR), examination of the mucin gene promoter, or in situ analysis Alternatively mucin proteins are assessed by conventional methodology, such as western blot analysis, ELISA, immunochemistry and the like, using detectably labeled antibodies Morphological criteria may also be used to determine the presence or absence of goblet cells in the culture, such as staining for mucins using Alcian blue/PAS staining (Lou et)
- in vivo animal models are used to assess the therapeutic potential of candidate agents to inhibit goblet cell proliferation
- the assay comprises the steps of (i) creating an animal model of hypersecretory pulmonary disease by inducing EGF-R expression, ( ) stimulating the induced EGF-R to produce mucin producing goblet cells, (in) treating with a candidate agent, and (iv) assessing goblet cell proliferation or mucus secretion, wherein an inhibition of goblet cell proliferation or mucus secretion is indicative of the candidate agent's therapeutic potential
- Any in vivo model of hypersecretory pulmonary disease may be used by way of example an asthmatic mouse model, as described in Temann ef al (1997) Am J Respir Cell Biol 16 471-478, and as shown in the examples provided herein Alternatively, a rat model can be used, as described by Takeyama ef al (1998) Am J Physiol Examples of other animal models that may be used include, but are not limited to Guinea pigs (a species that expresses goblet cells constitutively) and rats
- the lung tissue or tracheal tissue of the animal models may be assessed by the same molecular and biochemical markers described for the in vitro model A decrease in goblet cell proliferation is indicative of the therapeutic potential of the EGF-R antagonist
- Goblet cell hyperplasia occurs in various hypersecretory diseases of airways, but because the underlying mechanisms are unknown, no effective therapy exists In healthy airways, goblet cells are few, but in hypersecretory airway diseases, goblet cell hyperplasia occurs A human bronchial
- NCI-H292 tumor necrosis factor alpha
- instillation of TNF ⁇ , followed by EGF-R hgands resulted in an increased number of goblet and pre-goblet cells and a striking increase in Alcian blue/PAS-positive staining (reflecting mucous glycoconjugates) and mucin MUC5 gene expression
- ovalbumin resulted in goblet cell production and EGF-R expression in airway epithelium
- NCI-H292 cells in rats stimulated by TNF ⁇ followed by EGF-R hgands, and in the asthma model in rats, pretreatment with EGF-R tyrosine kinase inhibitor (BI)
- EGF fetal bovine serum
- penicillin 100 U/ml
- streptomycin 100 ⁇ g/ml
- cells were incubated with EGF (recombinant human EGF, 25 ng/ml, Genzyme, Cambridge, MA), TGF ⁇ (recombinant human TGF ⁇ , 25 ng/ml, Genzyme), TNF ⁇ (recombinant human TNF ⁇ , 20ng/ml, Genzyme), EGF (25 ng/ml) plus TNF ⁇ (20ng/ml) or TGF ⁇ (25 ng/ml) plus TNF ⁇ (20 ng/ml) for 12 h, 24 h or 48 h
- EGF-R tyrosine kinase inhibitor BIBX1522 (10 ⁇
- Triton X 1% sodium dioxycolate and PMSF (10 mg/ml) Total amount of protein was estimated by BCA protein assay reagent (Pierce, Rockford, IL) Cell lysates were boiled with Tricme sample buffer and 2% ⁇ ME at 95°C Proteins were separated by SDS-PAGE in 8% acrylamide gels The resulting gels were equilibrated in the transfer buffer 25 mM T ⁇ s-HCI, 192 mM glycme, 20% (vol/vol) methanol, pH 8 3 The proteins were then transferred electrophoretically to nitrocellulose membranes The membranes were then incubated for 1 h in 5% fat-free skim milk in PBS containing 0 05% Tween 20 Then the membranes were incubated with monoclonal mouse anti-EGF-R antibody (1 100) at 4°C overnight Bound antibody was visualized according to standard protocols for avidin-biotin-alkahne phosphatase complex method (ABC kit, Vector
- Probes EGF-R mRNA expression was determined using the linearized pTRI-EGF-R-human probe template (Ambion, Austin, TX) This probe contains a 360 bp cDNA fragment of the human EGF-R gene, which spans exons 12-14.
- MUC5 gene expression was determined using human MUC5AC probe, which contains a 298 bp cDNA fragment of human MUC5AC gene (generously provided by Dr. Carol Basbaum).
- Hybridization solution contained 250 mM Tris-HCl (pH7.5), 5% SDS, 1 % BSA, 1% polyvinyl-pyrrolidone, 1% Ficoll, and 0.5% sodium pyrophosphate. After hybridization, the membranes were washed twice with 2 x SSC with 0.1% SDS for 30 min at room temperature, followed by two washes in 2 x SSC with 0.1% SDS for 30 min at 50°C and a rinse in 0.1 x SSC with 0.1% SDS. Membranes were exposed to X-ray film.
- EGF 600 ng, 100 ⁇ l
- TGF ⁇ rat synthetic TGF ⁇ , 250 ng, 100 ⁇ l; Sigma, St Louis, Ml
- TNF ⁇ 200 ng, 100 ⁇ l
- sterile PBS 100 ⁇ l was instilled into the trachea as control.
- EGF-R tyrosine kinase inhibitor BIBX1522 (dose estimated from studies using the inhibitor to prevent cancer growth). Rats were pretreated with BIBX1522 (3, 10 or 30 mg/kg, i.p.), 1 h before and 24 h after instillation of TGF ⁇ . The trachea and lungs were removed for examination 48 h after the instillation of TGF ⁇ .
- Rats were sensitized on days 0 and 10 with intraperitoneal injections of ovalbumin (10 mg, grade V; Sigma, St. Louis, MO), complexed with 100 mg of aluminum hydroxide in 0.5 ml of sterile saline. Rats then rested for 10 days. On day 20, ovalbumin was delivered directly into the trachea; animals were challenged with 100 ⁇ l of 0.1% ovalbumin in saline by intratracheal instillation three times (days 20, 22 and 24). Rats were euthanized either without challenge (day 20), or 48 h after the third challenge (day 26). This procedure induced goblet cell metaplasia.
- sensitized rats were pretreated with an EGF-R tyrosine kinase inhibitor, BIBX1522.
- BIBX1522 10 mg/kg, i.p., 1 h before the challenge
- BIBX1522 was also instilled into the trachea together with ovalbumin (BIBX1522, 10 _5 M, 100 ⁇ l).
- BIBX1522 was also injected i.p. every 24 h until the day before the rats were euthanized. After the animals were euthanized, the trachea was removed 48 h after the third challenge.
- Tissue preparation At preselected times during anesthesia, the systemic circulation was perfused with 1% paraformaldehyde in DEPC-treated PBS at a pressure of 120 mmHg. The trachea was then removed and placed in 4% paraformaldehyde for 24 h. After fixation, trachea and lungs were embedded in either JB-4 plus monomer solution A for cell analysis or O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA) for immunohistochemistry and in situ hybridization. The embedded tissues were cut as cross sections (4 mm thick) and placed on slides.
- Goblet cells are tall, cuboidal, goblet to low columnar in shape, with abundant Alcian blue/PAS-stained granules filling most of the cytoplasm between the nucleus and the luminal surface.
- Pre-goblet cells are defined as cells with smaller mucus-stained areas ( ⁇ 1/3 height in epithelium from basement membrane to luminal surface) or with sparsely and lightly Alcian blue/PAS-stained, small granules.
- Ciliated cells are recognized by their ciliated borders, lightly stained cytoplasm, and large round nuclei.
- Non-granulated secretory cells are columnar in shape and extend from the lumen to the basal lamina.
- the cytoplasm stains light pink color, and a few tiny PAS-positive and Alcian blue-negative granules are observed in the cytoplasm.
- Basal cells are small flattened cells with a large nucleus, located just above the basal lamina but not reaching the airway lumen. Quantification of goblet cell production. Goblet cell production, was determined by the volume density of Alcian blue/PAS-stained mucosubstances on the mucosal surface epithelium using a semi-automatic imaging system described elsewhere (Weber ef al. (1984) Science 224:294-297).
- EGF-R Immunohistochemical localization of EGF-R in rat epithelium
- the localization of EGF-R was examined using immunohistochemical stammg with an antibody to EGF-R (Calbiochem, San Diego, CA) in frozen sections of rat trachea
- tissues were placed in 4% paraformaldehyde in PBS for 1 h and then removed in 30% sucrose for cryoprotection overnight Tracheas were embedded in O C T compound (Sakura Finetek U S A , Inc , Torrance, CA) and frozen Frozen sections (5 ⁇ m) were cut and placed on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) Immunostaining was performed similarly to the in vitro studies
- RNA probes for in situ hybridization A 320 bp cDNA fragment of rat MUC5 was subcloned into the Xba/hmdlll site of the transcription vector, pBluescr ⁇ pt-SK(-) (Stratagene, La Jolla, CA)
- this recombinant plasmid containing the rat MUC5 cDNA fragment was linearized and transcribed in vitro with the T7 or T3 polymerase to obtain antisense or sense probe, respectively
- the probes for in situ hybridization were generated in the presence of [ 35 S]UTP
- the cDNA template was digested with DNase, and radiolabeled RNA was purified via a Sephadex G-25 Quick SpinTM Column (Boehrmger Mannheim, Indianapolis, IN) and precipitated in an ethanol/ammonium acetate solution Before use, RNA probes were washed with 70% ethanol and diluted in 10 mM DTT
- NCI-H292 cells express EGF-R constitutively.
- Western analysis of immunoblots identified the presence of EGF-R protein in confluent cultures of NCI-H292 cells ( Figure 1A, right). Cells were examined after becoming confluent. Lysates were electrophoresed in 8% acrylamide gels and blotted with anti-EGF-R antibody. Molecular weights of marker proteins are reported on the right.
- a positive control for EGF-R was protein from A431 cells ( Figure 1A, left), which express EGF-R constitutively (Weber et al., supra.).
- Fig 1B Immunocytochemical analysis with anti-EGF-R antibody in cultures of NCI-H292 cells. At confluence, positive staining was seen, most strongly in dividing cells (arrows, right side). In the absence of the primary antibody, staining was absent (left side).
- Northern blotting showed that TNF ⁇ (20 ng/ml) up-regulated EGF-R gene expression, an effect that was present at 12 h and increased at 24 h (Fig. 1C, Northern analysis of EGF-R in NCI-H292 cells).
- EGF-R Ligands Stimulate Expression of Mucous Glycoconjugates and MUC5 Gene
- EGF-R are expressed constitutively in NCI-H292 cells, so we assessed the ability of EGF-R ligands (EGF, TGF ⁇ ) to induce the production of mucous glycoconjugates (Figure
- EGF-R tyrosine kinase inhibitor BIBX1522 (10 ⁇ g/ml). When cells were incubated alone (control), some PAS-positive staining was seen (arrows, upper column); incubation with TNF ⁇ (20 ng/ml) alone did not affect the staining; incubation with EGF; (25 ng/ml) or with TGF ⁇ (25 ng/ml) increased the
- NCI-H292 cells showed some expression in the control state (Figure 3, lower left column), when the cells were incubated with EGF or TGF ⁇ , MUC5 gene expression was barely recognized at 12 h but was clearly expressed at 24 h TNF ⁇ alone did not affect MUC5 gene expression, but when TNF ⁇ was added to the cells incubated with EGF-R hgands, MUC5 gene expression increased markedly above the level caused the EGF-R hgand alone ( Figure 3)
- EGF-R Tyrosine Kinase Inhibitor (BIBX1522) Prevents Expression of Mucous Glycoconjungates and of MUC5 Gene Expression in NCI-H292 Cells
- BIBX1522 EGF-R tyrosine kinase inhibitor
- FIG. 2 On Northern analysis, MUC5 gene expression that was markedly increased by the combination of TNF ⁇ plus EGF or plus TGF ⁇ was completely inhibited by pre-incubation with BIBX1522 ( Figure 3, lower column)
- tracheal epithelium contained few EGF-R-positive cells ( Figure 4A, left)
- intratracheal instillation of TNF ⁇ (200 ng) induced EGF-R protein in various cell types in the tracheal epithelium ( Figure 4A, right) EGF-R-positive staining was present in goblet cells (G), pre-goblet cells (P-G), non-granulated secretory cells (S), and basal cells (Ba), but not in ciliated cells
- TNF ⁇ induces EGF-R protein production Role of EGF-R Ligands in Production of Mucous Glycoconjugates and MUC5 Gene Expression in Rats
- tracheal epithelium contained few goblet and pre-goblet cells Intratracheal epithelium
- EGF-R Tyrosine Kinase Inhibitor (BIBX1522) Prevents Goblet Cell Production Induced by
- EGF-R when stimulated by EGF-R hgands, induce goblet cell production in vitro and in vivo, effects due to activation of EGF-R and which were blocked by an EGF-R tyrosine kinase inhibitor In an ovalbumin model of asthma, the inhibitor was also effective in preventing goblet cell production
- Asthma serves as an example of the therapeutic strategy of the invention
- Normal human airway epithelium has a ratio of 3-10 ciliated cells to each goblet cell
- the number of goblet cells can be equal or exceed ciliated cells, in patients who die in sfafivs asthmaticus, there is a 30-fold increase in the percentage area occupied by goblet cells compared with the number in patients dying of non-asthma respiratory diseases
- Inhibition of production of goblet cells should eliminate this source of hypersecretion
- the life cycle of goblet cells is unknown, the time course of resolution of goblet cell hyperplasia with treatment can not be predicted with precision
- Inhibition of EGF-R activation may inhibit goblet cell hyperplasia much more rapidly, depending on the life span of goblet cells
- Hypersecretion is a major manifestation in many chronic inflammatory diseases of airways
- Present findings provide a mechanism and a strategy for therapy by inhibiting EGF-R activation, goblet cell production is prevented
- Inhibitors of EGF-R activation are proposed as therapy in hypersecretory airway diseases
- Neutrophil isolation was performed by standard techniques of Ficoll-Hypaque gradient separation, dextran sedimentation, and ' hypotonic lysis of erythrocytes Cells were routinely
- NCI-H292 cells a human pulmonary mucoepidermoid carcinoma cell line, were grown in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and Hepes (25 mM) at 37°C in a humidified 5% C0 2 water-jacketed incubator Either 6- well culture plates or 8-chamber slides were used to culture the cells When confluent, cells were incubated for 1 h with neutrophils (10 6 cells/ml) alone, TNF ⁇ alone (recombinant human TNF ⁇ , 20ng/ml, Genzyme, Cambridge, MA), IL-8 (recombinant human IL-8, 10 8 M, Genzyme) alone, fMLP (10 8 M, Sigma, St Louis, MO) alone, TNF ⁇ plus neutrophils, IL-8 plus neutrophils, fMLP plus neutrophils, hydrogen peroxide (H2O2, 200 ⁇ M), cigarette smoke solution or TGF
- Cigarette smoke solution was prepared as previously described (Dusser et al (1989) J Clin Invest 84 900-906)
- MUC5AC mouse mAb to MUC5AC (clone 45 M1 , 1 200, Neo Markers, Fremont, CA) for 1 h at room temperature, and then washed 3 times with PBS to remove excess primary antibody
- TGF ⁇ protein was measured using a commercially available kit for ELISA (Sigma), following the manufacturer's instructions Supernatant taken after incubation of neutrophils plus TNF ⁇ (20 ng/ml) for 1 h was mixed with the lysis buffer PBS containing
- Triton X-100 1% Triton X-100, 1% sodium deoxycholate and several protease inhibitors, (Complete Mini, Boehrmger Mannheim, Germany), and then used to measure TGF ⁇
- EGF-R Tyrosine Kinase Inhibitors Prevent MUC5AC Synthesis Induced by Supernatant of Activated Neutrophils Because EGF-R hgands are known to cause MUC5AC synthesis in NCI-H292 cells via activation of EGF-R tyrosine kinase, the role of EGF-R activation in MUC5AC synthesis induced by the supernatant of activated neutrophils was examined Pretreatment of NCI-H292 cells with selective EGF-R tyrosine kinase inhibitors (BIBX1522, AG148), prevented the MUC5AC protein synthesis that was usually induced by the supernatant of activated neutrophils A selective platelet- derived growth factor receptor kinase inhibitor (AG1295) and a negative control for tyrphostins (A1) were without effect. These results implicate activation of EGF-R tyrosine kinase in MUC5AC synthesis induced by the
- EGF-R tyrosine kinase is dependent on the EGF-R hgands (EGF and TGF ⁇ ).
- EGF and TGF ⁇ EGF-R hgands
- Pretreatment of the supernatant with either anti-TGF ⁇ antibody or anti- EGF antibody did not inhibit MUC5AC synthesis induced by the supernatant of activated neutrophils
- TGF ⁇ was not detected in the supernatant
- EGF-R tyrosine phosphorylation caused by the supernatant of activated neutrophils was induced by a mechanism independent of the EGF-R hgands, EGF and TGF ⁇
- Cigarette smoke and the oxygen free radical, H 2 0 2 up-regulated MUC5AC gene expression within 12 h, as did TGF ⁇ Likewise, all stimuli increased MUC5AC protein synthesis and mucous glycoconjugate production within 24 h, effects that occurred in a dose-dependent fashion
- the maximum MUC5AC synthesis in response to H 2 0 2 was significantly less than the response to TGF ⁇
- Pretreatment with AG1478 prevented the increase in MUC5AC protein synthesis induced by all stimuli, indicating that the stimuli cause mucin synthesis by the activation of EGF-R tyrosine kinase MUC5AC synthesis by supernatant of activated neutrophils, cigarette smoke and H 2 0 2 were significantly inhibited by pretreatment with free radical scavengers (DMSO and DMTU) and SOD, but MUC5AC protein synthesis by TGF ⁇ was unaffected by DMSO or SOD In
- Agarose plugs Agarose plugs (0 7-0 8 mm diameter) were made with 4% agarose type II medium EEO (Sigma, St Louis, MO) in sterile phosphate-buffered saline (PBS) To visualize the agarose plugs in tissue, 3% suspension Monastral blue B (Sigma, St Louis, MO) was added after melting the agarose at 50°C
- mice were treated with BIBX1522 (80 mg/kg, i p ) 1 h before instillation of the agarose plugs and repeated daily (40 mg/kg, i p , bid) Animals were euthanized 24, 48 or 72 h after instillation of the agarose plugs.
- animals were treated with a TNF ⁇ neutralizing antibody (Genzyme, Boston, MA) The first treatment (100 ⁇ l in 0 2 ml saline, i p ) was given 1 h before the instillation of agarose plugs, and i p injections were repeated daily
- TNF ⁇ neutralizing antibody was infused (10 ⁇ l/h) via an osmotic minipump (Alzet 2ML1 , Alza Corp , Palo Alto, CA) implanted
- All drugs (BIBX1522, TNF ⁇ neutralizing antibody, cyclophosphamide, and NPC 15669) were given i p 1 h before instillation of agarose plugs, and doses were repeated daily for 3 d Tissue preparation.
- rats were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the systemic circulation was perfused with 1% paraformaldehyde in diethyl pyrocarbonate-treated PBS at a pressure of 120 mmHg.
- the right lung was removed, and the right caudal lobe was used for histology.
- tissues were removed and placed in 4% paraformaldehyde for 1 h and then replaced in 30% sucrose for cryoprotection overnight.
- the tissues were embedded in O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA).
- methacrylate sections the tissues were placed in 4% paraformaldehyde for 24 h and then dehydrated with graded concentrations of ethanol and embedded in methacrylate JB-4 (Polysciences, Inc., Warrington, PA). Tissue sections (4 ⁇ m thick) were stained with Alcian blue/PAS and counterstained with hematoxylin.
- Morphometric analysis of bronchial epithelium The percentage of Alcian blue/PAS-stained area of mucous glycoconjugates in the epithelium was determined using a semi-automatic image analysis system according to previously published methods. The area of epithelium and Alcian blue/PAS-stained mucous conjugates within the epithelium was manually circumscribed and analyzed using the NIH Image program (developed at the U.S. National Institutes of health and available from the internet by ananymous FTP or from a floppy disk from the National Technical Information Service, Springfield, Virginia; part number PB95-500195GEI). The data are expressed as the percentage of the total epithelial area occupied by Alcian blue/PAS stain.
- the percentage of the length of epithelial surface occupied by Alcian blue/PAS-positive staining was determined by calculating the length that stained positively as a ratio to the total length.
- the percentage of denuded epithelium was determined by calculating the ratio of the length of denuded epithelium to the total epithelial length.
- the total number of epithelial cells was determined by counting epithelial cell nuclei over 2 mm of the basal lamina with an oil immersion objective lens (x1000 magnification).
- the linear length of the basal lamina under each analyzed region of epithelium was determined by tracing the contour of the digitalized image of the basal lamina.
- the epithelial cells were identified as described previously. In brief, basal cells were identified as small flattened cells with a large nucleus, located just above the basal lamina but not reaching the airway lumen. The cytoplasm stained darkly, and Alcian blue or PAS-positive granules were not present.
- Ciliated cells were recognized by their ciliated borders, lightly stained cytoplasm, and large, round nucleus.
- Non-granulated secretory cells were columnar in shape and extended from the bronchial lumen to the basal lamina. After intrabronchial instillation of agarose plugs, "developing" goblet cells (pre-goblet cells) were formed.
- EGF-R Immunohistochemical localization of EGF-R The presence of EGF-R was determined by immunohistochemical localization, using a monoclonal mouse antibody to the EGF-R (Calbiochem, San Diego).
- Biotinylated horse anti-mouse IgG (1 200, Vector Lab , Burhngame, CA), followed by streptavidin- peroxidase complex (ABC kit, Vector Lab , Burhngame, CA) was used to visualize antigen-antibody complexes stained with 3, 3'-d ⁇ am ⁇ nobenz ⁇ d ⁇ ne tetrahydrochlonde (Sigma, St Louis, MO) Negative control slides were incubated with either the primary or secondary antibody omitted and replaced with
- Bronchoalveolar lavage(BAL) To assess differential cell counts in each group of animals, lungs were lavaged five times with 3 ml aliquots of sterile PBS, lavages were pooled, and the volume was measured Cells in BAL were collected by spinning the lavage fluid at 1,000 rpm for 10 m Ten microliters of a cell suspension was then counted with a hematocytometer to determine cell numbers in BAL fluid Differential cell counts were performed on cytospun preparations stained with Diff-Quik (American Scientific Products, McGaw Park, IL) Differential cell counts were obtained by sampling at least 200 cells on each cytospun slide
- Effecf of agarose plugs on EGF-R expression in airway epithelium In control animals, immunostainmg with an antibody to EGF-R showed sparse staining in epithelium However, after instillation of agarose plugs, epithelium adjacent to agarose plugs showed EGF-R-positive staining in cells that stained positively with Alcian blue/PAS The staining pattern for EGF-R paralleled the staining for MUC5AC and AB/PAS Pre-goblet, goblet, and non-granulated secretory cells were immunopositive for EGF-R Ciliated cells showed no immunoreactivity In airways not obstructed by agarose plugs, the epithelium showed little staining for EGF-R and appeared similar to staining in control animals Effecf of EGF-R tyrosine kinase inhibitor on goblet cell metaplasia and on mucin gene expression.
- EGF-R is a member of the class of tyrosine kinase receptors.
- EGF-R ligands EGF or TGF ⁇
- a specific EGF-R tyrosine kinase is activated. Therefore, to test the hypothesis that EGF-R activation induces expression of MUC5AC gene and of mucous glycoconjugates after instillation of agarose plugs, an EGF-R tyrosine kinase inhibitor (BIBX1522) was injected intrapentoneally in rats.
- BIBX1522 markedly inhibited agarose plug-induced Alcian blue/PAS-stained area of epithelium at 24, 48 and 72 h. It also completely inhibited the expression of MUC5AC gene at 72 h after plug instillation.
- Infiltrating cells were also evaluated in tissue sections: airways without agarose plugs contained few neutrophils, but airways containing plugs showed presence of neutrophils, both in the epithelium and in the lumen.
- EGF-R are not normally expressed in airway epithelium of pathogen-free rats but is induced by TNF ⁇ .
- EGF-R ligands EGF or TGF ⁇
- a selective inhibitor of EGF-R tyrosine kinase (BIBX1522) completely inhibits these responses, implicating EGF-R signaling in goblet cell metaplasia.
- BIBX1522 inhibited agarose plug-induced production of mucous glycoconjugates and MUC5AC gene expression. These results implicate an EGF-R cascade in agarose plug-induced goblet cell metaplasia.
- Cyclophosphamide a drug that selectively depresses leukocyte production, prevented neutrophil recruitment into airway lavage fluid and into the airway epithelium following the instillation of agarose plugs and also prevented agarose plug-induced goblet cell metaplasia. Macrophages were also increased after the introduction of agarose plugs, but cyclophosphamide did not inhibit macrophage recruitment. These results implicate neutrophils in agarose plug-induced goblet cell metaplasia. The fact that cyclophosphamide also decreased EGF-R protein expression after agarose plugs suggests that neutrophils contribute, at least in part, to the EGF-R expression in this inflammatory condition.
- Neutrophils are also capable of producing the EGF-R ligands, EGF and TGF ⁇ .
- epithelial cells are sources of EGF-R ligands, and there was striking denudation of epithelium adjacent to the agarose plugs.
- the epithelium could be an important potential source of both TNF ⁇ and EGF-R ligands.
- Epithelial damage is a common finding in studies of patients even with mild asthma, and the damage is increasingly related to worsening of clinical symptoms.
- Epithelial damage produced by the allergic response may induce EGF-R activation, which results in abnormal goblet cell production.
- the data presented above implicate EGF-R activation in a different response, specifically involving goblet cell metaplasia.
- Mechanical epithelial damage and epithelial injury in asthma may involve a similar (EGF-R) cascade, resulting in abnormal growth of epithelial secretory cells. This provides a mechanism for the hypersecretion that occurs in fatal cases of acute asthma.
- Example 4 Regranulation of goblet cell by EGF- Receptors Degranulation of goblet cells in rat nasal respiratory epithelium was induced by infranasal inhalation of fMLP. Significant degranulation was induced in the nasal septal epithelium 4 h after infranasal inhalation of fMLP (10 "7 M). Goblet cell regranulation occurred by 48 h after inhalation. In the control state, MUC5AC protein was expressed in the goblet cells, but EGF-R protein was not expressed. Both EGF-R and MUC5AC mucin gene and protein were absent in control epithelium but were expressed significantly 48 h after inhalation.
- EGF-R tyrosine kinase inhibitor BIBX1522
- the head was decalcified with Surgipath (Decalcifier II, Surgical Medical Industries, Inc., Richmond, IL) for 4 - 5 days and rinsed in phosphate- buffered saline.
- Surgipath Decalcifier II, Surgical Medical Industries, Inc., Richmond, IL
- the nasal cavity was sectioned transversely at the level of the incisive papilla of the nasal palate.
- the frontal tissue block was embedded in glycol methacrylate (JB 4 Plus, Polysciences, Inc., Warrington, PA), or in OCT compound (Sakura Finetek, U.S.A., Inc., Torrance, CA) for frozen sections.
- Neutrophils were counted in high power fields of the epithelial layer stained with 3,3'-diaminobenzidine at magnification x400. The number of neutrophils within the nasal sepal epithelium (from the basement membrane to cell apices) was determined by counting the number of nuclear profiles per unit of basal lamina length.
- the intracellular mucin in superficial epithelial secretory cells appears as oval-shaped, purple granules of varying sizes.
- EGF-R and MUC5AC protein Immunolocalization of EGF-R and MUC5AC protein. Frozen sections from the paraformaldehyde-fixed nasal tissues were treated with 3 % H 2 0 2 /methanol to block endogenous peroxide and were incubated with a mouse monoclonal antibody to EGF-R (Calbiochem, San Diego, CA), or MUC5AC (NeoMarkers Inc., Fremont, CA) for 1 h at a dilution of 1 :100. Immunoreactive EGF- R or MUC5AC was visualized with the Vectastain Elite ABC kit (Vector Lab., Inc., Burhngame, CA) using 3,3-diaminobenzidine tetrahydrochlonde as a chromogen.
- Vectastain Elite ABC kit Vector Lab., Inc., Burhngame, CA
- Controls included the substitution of primary or secondary antibody with PBS.
- Methods We studied pathogen-free rats, which normally have many goblet cells in the nasal septal epithelium. To determine the effect of aerosolized fMLP on goblet cell degranulation and on neutrophil migration into nasal mucosa epithelium, the animals were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), and they received N-formyl-methionyl-leucyl-phenylalanine (fMLP; 10 ⁇ 5 M, Sigma, St. Louis, MO) in pyrogen-free saline intranasally by aerosol for 5 min.
- fMLP N-formyl-methionyl-leucyl-phenylalanine
- Aerosol exposure was accomplished by ventilating the animals with an ultrasonic nebulizer (PulmoSonic, DeVilbiss Co., Somerset, PA) that generated an aerosol mist at rate of 0.3 ml/min. Similarly, control animals were given saline aerosol alone intranasally.
- an ultrasonic nebulizer PulmoSonic, DeVilbiss Co., Somerset, PA
- EGF-R tyrosine kinase activation was evaluated on goblet cell re-granulation.
- animals were pretreated intrapentoneally with an EGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg, generously provided by Boehringer Ingelheim Inc., Ingelheim, Germany) 30 min before the inhalation of fMLP and repeated twice a day.
- an EGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg, generously provided by Boehringer Ingelheim Inc., Ingelheim, Germany) 30 min before the inhalation of fMLP and repeated twice a day.
- non-stimulated rat nasal epithelium contains "stable", non- degranulating goblet cells containing mucin proteins.
- Neutrophil chemoattractants e.g., fMLP
- fMLP neutrophil chemoattractants
- EGF-R EGF-receptors
- EGF-R In pathogen-free rats goblet cells are "inactive" (ie, not degranulating) and EGF-R are down-regulated. When inflammation (eg, stimulation of neutrophil infiltration) causes GC degranulation and mucin secretion, up-regulation and activation of EGF-R re-supplies the airway epithelium with mucins.
- inflammation eg, stimulation of neutrophil infiltration
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Pulmonology (AREA)
- Environmental Sciences (AREA)
- Epidemiology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Otolaryngology (AREA)
- Mycology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Priority Applications (21)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IL14085599A IL140855A0 (en) | 1998-08-18 | 1999-08-17 | Preventing airway mucus production by administration of egf-r antagonists |
DE69939022T DE69939022D1 (de) | 1998-08-18 | 1999-08-17 | Epidermale wachstumsfaktor rezeptor antagonisten zur behandlung stark vermehrter schleimsekretion in der lunge |
SK216-2001A SK287805B6 (sk) | 1998-08-18 | 1999-08-17 | Použitie antagonistu receptora epidermálneho rastového faktora (EGF-R) na výrobu lieku na liečenie nadmerného vylučovania hlienu v dýchacích cestách |
PL351765A PL200940B1 (pl) | 1998-08-18 | 1999-08-17 | Zastosowanie antagonisty receptora naskórkowego czynnika wzrostowego (EGF-R) i postać farmaceutyczna do leczenia nadmiernego wydzielania śluzu dróg oddechowych |
DK99943729T DK1119349T3 (da) | 1998-08-18 | 1999-08-17 | Epidermal vækstfaktor receptorantagonister til behandling af voldsomt foröget slimudskillelse i lungerne |
SI9931016T SI1119349T1 (sl) | 1998-08-18 | 1999-08-17 | Antagonisti receptorja epidermalnega rastnega faktorja za zdravljenje hipersekrecije sluzi v pljučah |
CA2337422A CA2337422C (fr) | 1998-08-18 | 1999-08-17 | Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r |
UA2001021134A UA73722C2 (en) | 1998-08-18 | 1999-08-17 | Treatment of mucus hypersecretion in lungs by administration of epidermal growth factor receptor (egf-r) antagonist and pharmaceutical formulation |
BR9912672-9A BR9912672A (pt) | 1998-08-18 | 1999-08-17 | Prevenção de produção de muco no trato respiratório por administração de antagonistas de egf-r |
HU0103894A HUP0103894A3 (en) | 1998-08-18 | 1999-08-17 | Preventing airway mucus production by administration of egf-r antagonists |
JP2000565908A JP2002539072A (ja) | 1998-08-18 | 1999-08-17 | Egf−rアンタゴニストの投与による気道粘液産生の防止 |
EP99943729A EP1119349B1 (fr) | 1998-08-18 | 1999-08-17 | Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r |
AU56766/99A AU760766B2 (en) | 1998-08-18 | 1999-08-17 | Preventing airway mucus production by administration of EGF-R antagonists |
EEP200100097A EE200100097A (et) | 1998-08-18 | 1999-08-17 | Hingamisteede lima tootmise tõkestamine EGF-R antagonistide manustamise teel |
NZ509271A NZ509271A (en) | 1998-08-18 | 1999-08-17 | Epidermal growth factor receptor antagonists for treating hypersecretion of mucus in the lungs |
EA200100136A EA004316B1 (ru) | 1998-08-18 | 1999-08-17 | Способ лечения гиперсекреции слизи |
IL140855A IL140855A (en) | 1998-08-18 | 2001-01-10 | Use of an epidermal growth factor receptor (egf-r) antagonist for the preparation of pharmaceutical formulation for the treatment of airway mucus hypersecretion |
BG105158A BG105158A (bg) | 1998-08-18 | 2001-01-16 | Предотвратяване образуването на слузести покрития(мускус) върху летателни писти с прилагане на противодействащо средство egf-r |
NO20010749A NO327000B1 (no) | 1998-08-18 | 2001-02-14 | Anvendelse av epidermal vekstfaktorreseptor (EGF-R) antagonister |
HR20010119A HRP20010119B1 (en) | 1998-08-18 | 2001-02-16 | Preventing airway mucus production by administration of egf-r antagonists |
HK01106832A HK1036219A1 (en) | 1998-08-18 | 2001-09-27 | Epidermal growth factor receptor antagonists for treating hypersecretion of mucus in the lungs |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9702398P | 1998-08-18 | 1998-08-18 | |
US60/097,023 | 1998-08-18 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2000010588A2 true WO2000010588A2 (fr) | 2000-03-02 |
WO2000010588A9 WO2000010588A9 (fr) | 2000-08-10 |
WO2000010588A3 WO2000010588A3 (fr) | 2001-05-25 |
Family
ID=22260380
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/018696 WO2000010588A2 (fr) | 1998-08-18 | 1999-08-17 | Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r |
Country Status (36)
Country | Link |
---|---|
US (6) | US6270747B1 (fr) |
EP (2) | EP1970058A1 (fr) |
JP (2) | JP2002539072A (fr) |
KR (1) | KR100609646B1 (fr) |
CN (1) | CN1184961C (fr) |
AR (1) | AR020220A1 (fr) |
AT (1) | ATE399541T1 (fr) |
AU (1) | AU760766B2 (fr) |
BR (1) | BR9912672A (fr) |
CA (1) | CA2337422C (fr) |
CO (1) | CO5130028A1 (fr) |
CY (1) | CY1110370T1 (fr) |
CZ (1) | CZ2001584A3 (fr) |
DE (1) | DE69939022D1 (fr) |
DK (1) | DK1119349T3 (fr) |
EA (1) | EA004316B1 (fr) |
EE (1) | EE200100097A (fr) |
ES (1) | ES2310047T3 (fr) |
HK (1) | HK1036219A1 (fr) |
HR (1) | HRP20010119B1 (fr) |
HU (1) | HUP0103894A3 (fr) |
ID (1) | ID27345A (fr) |
IL (2) | IL140855A0 (fr) |
MY (1) | MY126490A (fr) |
NO (1) | NO327000B1 (fr) |
NZ (1) | NZ509271A (fr) |
PH (2) | PH12010000215A1 (fr) |
PL (1) | PL200940B1 (fr) |
PT (1) | PT1119349E (fr) |
RS (1) | RS50273B (fr) |
SI (1) | SI1119349T1 (fr) |
SK (1) | SK287805B6 (fr) |
TR (1) | TR200100560T2 (fr) |
TW (1) | TWI250019B (fr) |
UA (1) | UA73722C2 (fr) |
WO (1) | WO2000010588A2 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002005842A3 (fr) * | 2000-07-14 | 2003-01-03 | Univ California | Administration d'antagonistes d'egf visant a empecher la production de mucus dans les voies respiratoires |
US6551989B2 (en) | 1998-08-18 | 2003-04-22 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US7354894B2 (en) | 1998-08-18 | 2008-04-08 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
Families Citing this family (45)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030149113A1 (en) * | 2001-10-12 | 2003-08-07 | Caplan Michael J. | Conductance of improperly folded proteins through the secretory pathway and related methods for treating disease |
US20030236300A1 (en) * | 1999-10-27 | 2003-12-25 | Yale University | Conductance of improperly folded proteins through the secretory pathway and related methods for treating disease |
WO2002094318A1 (fr) * | 2001-05-18 | 2002-11-28 | University Of Southern California | Vecteur d'apport cible aux cellules |
US6818446B2 (en) | 2001-11-21 | 2004-11-16 | The Regents Of The University Of California | Compositions and methods for the analysis of mucin gene expression and identification of drugs having the ability to inhibit mucin gene expression |
DE10204462A1 (de) * | 2002-02-05 | 2003-08-07 | Boehringer Ingelheim Pharma | Verwendung von Tyrosinkinase-Inhibitoren zur Behandlung inflammatorischer Prozesse |
US6924285B2 (en) * | 2002-03-30 | 2005-08-02 | Boehringer Ingelheim Pharma Gmbh & Co. | Bicyclic heterocyclic compounds, pharmaceutical compositions containing these compounds, their use and process for preparing them |
WO2003090681A2 (fr) * | 2002-04-24 | 2003-11-06 | Research Development Foundation | Effets synergetiques des inhibiteurs du facteur nf-kb de transcription nucleaire et d'agents anti-cancereux |
AU2003253681B2 (en) * | 2002-06-24 | 2008-05-01 | Research Development Foundation | Treatment of human multiple myeloma by Curcumin |
US20060241130A1 (en) * | 2003-01-31 | 2006-10-26 | Ehud Keinan | Anti-inflammatory compositions and uses thereof |
US7195899B1 (en) * | 2003-03-14 | 2007-03-27 | Florida State University Research Foundation, Inc. | Cell-based biosensor for harmful airborne agents |
CA2536738A1 (fr) * | 2003-08-26 | 2005-03-10 | Research Development Foundation | Administration par aerosol de curcumine |
AU2004320907A1 (en) | 2003-08-26 | 2006-04-13 | Research Development Foundation | Osteoclastogenesis inhibitors and uses thereof |
US11324698B2 (en) | 2003-08-28 | 2022-05-10 | Vgsk Technologies, Inc. | Sterically stabilized carrier for aerosol therapeutics, compositions and methods for treating the respiratory tract of a mammal |
US20060115523A1 (en) * | 2004-12-01 | 2006-06-01 | Konduri Kameswari S | Sterically stabilized liposome and triamcinolone composition for treating the respiratory tract of a mammal |
US8846079B1 (en) | 2004-12-01 | 2014-09-30 | Vgsk Technologies, Inc. | Sterically stabilized carrier for aerosol therapeutics, compositions and methods for treating the respiratory tract of a mammal |
US20050096332A1 (en) * | 2003-10-30 | 2005-05-05 | Boehringer Ingelheim International Gmbh | Use of tyrosine kinase inhibitors for the treatment of inflammatory processes |
EP2251357A1 (fr) | 2003-11-07 | 2010-11-17 | Ablynx N.V. | Anticorps VHH de domaine unique de camélidés dirigés contre le récepteur du facteur de croissance épidermique et utilisations correspondantes |
US8134010B2 (en) * | 2004-05-05 | 2012-03-13 | Renopharm Ltd. | Thiazole-based nitric oxide donors having aryl substituent(s) and uses thereof |
US7498445B2 (en) * | 2004-05-05 | 2009-03-03 | Renopharm Ltd. | Thiazole-based nitric oxide donors capable of releasing two or more nitric oxide molecules and uses thereof |
EP1753734A1 (fr) * | 2004-05-05 | 2007-02-21 | Renopharm Ltd. | Donneurs d'oxyde nitrique et utilisations de ceux-ci |
US7968575B2 (en) * | 2004-05-05 | 2011-06-28 | Renopharm Ltd. | Nitric oxide donors and uses thereof |
US6998028B1 (en) * | 2004-09-24 | 2006-02-14 | Superpower, Inc. | Methods for forming superconducting conductors |
EP1793837A2 (fr) * | 2004-09-27 | 2007-06-13 | Technion Research & Development Foundation Limited | Polylysines modifiees par l'acide gras comme agents microbicides |
US7915223B2 (en) * | 2004-09-27 | 2011-03-29 | Technion Research & Development Foundation Ltd. | Antimicrobial agents |
US20080072338A1 (en) * | 2004-09-28 | 2008-03-20 | Fuji Biomedix Co., Ltd | Animal for Drug Efficacy Evaluation, Method for Developing Chronic Obstructive Pulmonary Disease in Animal for Drug Efficacy Evaluation, and Method for Evaluating Drug Efficacy Using the Animal |
JP4690158B2 (ja) * | 2004-09-28 | 2011-06-01 | スギ生物科学研究所株式会社 | 薬効評価用動物、薬効評価用動物の慢性閉塞性肺疾患発症方法及びその動物を用いた薬効評価方法 |
JP2009511586A (ja) * | 2005-10-11 | 2009-03-19 | ワシントン・ユニバーシティ | 気道分泌過多を治療するための組成物および方法 |
EP1921070A1 (fr) * | 2006-11-10 | 2008-05-14 | Boehringer Ingelheim Pharma GmbH & Co. KG | heterocycles bicycliques, medicaments á base de ces composes, leur usage et procédé pour leur preparation |
MX2009007610A (es) * | 2007-02-06 | 2009-07-24 | Boehringer Ingelheim Int | Heterociclicos biciclicos, medicamentos que contienen estos compuestos, su utilizacion y procedimientos para su preparacion. |
US20080293740A1 (en) * | 2007-04-03 | 2008-11-27 | Parion Sciences, Inc. | Method of treating acid-sensing ion channel mediated pain, cough suppression, and central nervous system disorders |
EP2152289A1 (fr) * | 2007-04-30 | 2010-02-17 | Technion Research & Development Foundation Ltd. | Nouveaux agents antimicrobiens |
US20100173832A1 (en) * | 2007-04-30 | 2010-07-08 | Technion Research & Development Foundation Ltd. | Anticancerous polymeric agents |
WO2009098061A1 (fr) * | 2008-02-07 | 2009-08-13 | Boehringer Ingelheim International Gmbh | Hétérocycles spirocycliques, médicaments contenant ces composés, leur utilisation et procédé pour les produire |
JP5539351B2 (ja) * | 2008-08-08 | 2014-07-02 | ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング | シクロヘキシルオキシ置換ヘテロ環、これらの化合物を含有する医薬、およびそれらを生成するための方法 |
US8112365B2 (en) * | 2008-12-19 | 2012-02-07 | Foster Scott C | System and method for online employment recruiting and evaluation |
WO2010085665A2 (fr) | 2009-01-23 | 2010-07-29 | Cedars-Sinai Medical Center | Système d'administration ciblée |
US8597695B1 (en) | 2010-11-13 | 2013-12-03 | Sirbal Ltd. | Herbal combinations for treatment of a skin condition |
US8541382B2 (en) | 2010-11-13 | 2013-09-24 | Sirbal Ltd. | Cardiac glycoside analogs in combination with emodin for cancer therapy |
US9066974B1 (en) | 2010-11-13 | 2015-06-30 | Sirbal Ltd. | Molecular and herbal combinations for treating psoriasis |
US9095606B1 (en) | 2010-11-13 | 2015-08-04 | Sirbal Ltd. | Molecular and herbal combinations for treating psoriasis |
CN103732611B (zh) * | 2011-01-10 | 2016-06-01 | 伊沃恩有限公司 | β-肾上腺素能反向激动剂用于戒烟的用途 |
JP6618360B2 (ja) | 2012-08-03 | 2019-12-11 | セダーズ−シナイ メディカル センター | 薬物送達タンパク質の輸送向上変異体の単離 |
WO2015017728A1 (fr) | 2013-07-31 | 2015-02-05 | Windward Pharma, Inc. | Composés inhibiteurs de tyrosine kinase en aérosol et leurs utilisations |
EP3498288A1 (fr) | 2013-12-02 | 2019-06-19 | Sirbal Ltd. | Combinaisons de plantes pour le traitement d'une affection cutanée |
EP3328498B1 (fr) | 2015-07-29 | 2021-05-05 | Sirbal Ltd. | Kit medicale comprenant des combinaisons à base d'herbes permettant de traiter le psoriasis |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0157284A2 (fr) * | 1984-04-02 | 1985-10-09 | CAMILLO CORVI S.p.A. | Ester théophylline-7-acide acétique de D,L-trans-sobrérol ayant une activité mucosécrétolytique-fluidifiant et antibronchospastique, procédé pour sa préparaton et ses compositions pharmaceutiques |
WO1990003374A1 (fr) * | 1988-09-28 | 1990-04-05 | The Upjohn Company | 1,4-dihydrothionapthoquinone et congeneres heterocycliques qui inhibent les enzymes lipoxygenase |
WO1997019065A1 (fr) * | 1995-11-20 | 1997-05-29 | Celltech Therapeutics Limited | 2-anilinopyrimidines substituees utiles en tant qu'inhibiteurs de proteine kinase |
WO1999032121A1 (fr) * | 1997-12-19 | 1999-07-01 | Smithkline Beecham Corporation | .0mposes d'imidazole heteroaryle substitues, leurs compositions et usages pharmaceutiques |
Family Cites Families (57)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2932481A (en) * | 1954-06-15 | 1960-04-12 | Breer | Bipod camera support |
GB1242211A (en) * | 1967-08-08 | 1971-08-11 | Fisons Pharmaceuticals Ltd | Pharmaceutical composition |
FR2502627A1 (fr) * | 1981-02-02 | 1982-10-01 | Refarmed Sa | Derives thioliques de l'erythromycine a activite therapeutique, procede de preparation et produits pharmaceutiques dans lesquels ils apparaissent |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4904786A (en) * | 1986-01-27 | 1990-02-27 | American Home Products Corporation | Quinoline compounds as antiallergic and antiinflammatory agents |
US5089516A (en) * | 1987-03-11 | 1992-02-18 | Kanegafuchi Kagaku Kogyo Kabushiki Kaisha | 1-phenyl-3,5-pyrazolidinedione hydroxystyrene compounds which have tyrosine kinase inhibiting activity |
US5217999A (en) | 1987-12-24 | 1993-06-08 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Styryl compounds which inhibit EGF receptor protein tyrosine kinase |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
GB8904009D0 (en) | 1989-02-22 | 1989-04-05 | Celltech Ltd | Vector |
US5362755A (en) * | 1990-01-05 | 1994-11-08 | Sepracor, Inc. | Method for treating asthma using optically pure (R)-albuterol |
US5302606A (en) * | 1990-04-16 | 1994-04-12 | Rhone-Poulenc Rorer Pharmaceuticals Inc. | Styryl-substituted pyridyl compounds which inhibit EGF receptor tyrosine kinase |
US5165424A (en) | 1990-08-09 | 1992-11-24 | Silverman Harvey N | Method and system for whitening teeth |
US5404871A (en) | 1991-03-05 | 1995-04-11 | Aradigm | Delivery of aerosol medications for inspiration |
CZ282603B6 (cs) * | 1991-03-06 | 1997-08-13 | Merck Patent Gesellschaft Mit Beschränkter Haftun G | Humanizované a chimerické monoklonální protilátky |
US5116616A (en) * | 1991-04-29 | 1992-05-26 | Bio-Technology General Corp. | Use of intratracheal administration of SOD to protect humans from lung injury due to hyperoxia and hyperventilation |
DE4117078A1 (de) * | 1991-05-25 | 1992-11-26 | Boehringer Ingelheim Kg | Verfahren zur herstellung therapeutisch anwendbarer aerosole |
DE69233690T2 (de) | 1991-07-02 | 2008-01-24 | Nektar Therapeutics, San Carlos | Abgabevorrichtung für nebelförmige Medikamente |
PT100905A (pt) * | 1991-09-30 | 1994-02-28 | Eisai Co Ltd | Compostos heterociclicos azotados biciclicos contendo aneis de benzeno, ciclo-hexano ou piridina e de pirimidina, piridina ou imidazol substituidos e composicoes farmaceuticas que os contem |
DE4310051A1 (de) | 1992-04-11 | 1993-10-14 | Byk Gulden Lomberg Chem Fab | Weitere Verwendung substituierter Pyridazine |
US5785049A (en) | 1994-09-21 | 1998-07-28 | Inhale Therapeutic Systems | Method and apparatus for dispersion of dry powder medicaments |
WO1994006283A1 (fr) * | 1992-09-11 | 1994-03-31 | The Regents Of The University Of Michigan | Animal autre que l'homme possedant une heterogreffe sur un conduit respiratoire peuple par des cellules humaines |
IL108367A0 (en) * | 1993-01-27 | 1994-04-12 | Hektoen Inst For Medical Resea | Antisense polynzcleotide inhibition of human growth factor-sensitive cancer cells |
US5497763A (en) | 1993-05-21 | 1996-03-12 | Aradigm Corporation | Disposable package for intrapulmonary delivery of aerosolized formulations |
GB9314884D0 (en) | 1993-07-19 | 1993-09-01 | Zeneca Ltd | Tricyclic derivatives |
GB9314893D0 (en) | 1993-07-19 | 1993-09-01 | Zeneca Ltd | Quinazoline derivatives |
US5716981A (en) * | 1993-07-19 | 1998-02-10 | Angiogenesis Technologies, Inc. | Anti-angiogenic compositions and methods of use |
WO1995006764A2 (fr) | 1993-09-03 | 1995-03-09 | Vpi Holdings Ltd. | Oligonucleotides ayant une activite de segmentation de l'arn |
EP0719412B1 (fr) * | 1993-09-13 | 1998-11-04 | Merck Frosst Canada Inc. | Methode rapide d'evaluation des modifications metaplasiques de cellules epitheliales secretrices de mucus |
CA2261433A1 (fr) * | 1993-12-09 | 1995-06-10 | Belinda Sanchez Ramirez | Composition comprenant un facteur de croissance epidermique autologue |
AU706417B2 (en) | 1994-02-23 | 1998-06-17 | Ribozyme Pharmaceuticals, Inc. | Method and reagent for inhibiting the expression of disease related genes |
AU2096895A (en) | 1994-03-07 | 1995-09-25 | Sugen, Incorporated | Receptor tyrosine kinase inhibitors for inhibiting cell proliferative disorders and compositions thereof |
US20030114379A1 (en) * | 1994-03-08 | 2003-06-19 | Human Genome Sciences, Inc. | Methods of treating or preventing cell, tissue, and organ damage using human myeloid progenitor inhibitory factor-1 (MPIF-1) |
GB9510757D0 (en) * | 1994-09-19 | 1995-07-19 | Wellcome Found | Therapeuticaly active compounds |
EP3103799B1 (fr) | 1995-03-30 | 2018-06-06 | OSI Pharmaceuticals, LLC | Derives de quinazoline |
EP0824525B1 (fr) | 1995-04-27 | 2001-06-13 | AstraZeneca AB | Derives de quinazoline |
GB9508565D0 (en) | 1995-04-27 | 1995-06-14 | Zeneca Ltd | Quiazoline derivative |
GB9508538D0 (en) | 1995-04-27 | 1995-06-14 | Zeneca Ltd | Quinazoline derivatives |
US5747498A (en) * | 1996-05-28 | 1998-05-05 | Pfizer Inc. | Alkynyl and azido-substituted 4-anilinoquinazolines |
US5760041A (en) * | 1996-02-05 | 1998-06-02 | American Cyanamid Company | 4-aminoquinazoline EGFR Inhibitors |
IL126351A0 (en) * | 1996-04-12 | 1999-05-09 | Warner Lambert Co | Irreversible inhibitors of tyrosine kinases |
ES2231880T3 (es) * | 1996-07-29 | 2005-05-16 | Paringenix, Inc. | Procedimiento para tratar el asma con heparina o-desulfatada. |
SE9603725D0 (sv) | 1996-10-11 | 1996-10-11 | Astra Ab | New teatment |
UA73073C2 (uk) | 1997-04-03 | 2005-06-15 | Уайт Холдінгз Корпорейшн | Заміщені 3-ціанохіноліни, спосіб їх одержання та фармацевтична композиція |
ZA986729B (en) | 1997-07-29 | 1999-02-02 | Warner Lambert Co | Irreversible inhibitors of tyrosine kinases |
ZA986732B (en) | 1997-07-29 | 1999-02-02 | Warner Lambert Co | Irreversible inhibitiors of tyrosine kinases |
TW436485B (en) | 1997-08-01 | 2001-05-28 | American Cyanamid Co | Substituted quinazoline derivatives |
US6251912B1 (en) * | 1997-08-01 | 2001-06-26 | American Cyanamid Company | Substituted quinazoline derivatives |
DE69940808D1 (de) * | 1998-03-04 | 2009-06-10 | Bristol Myers Squibb Co | Heterocyclen substituierte imidazopyrazine als protein- tyrosin-kinase-inhibitoren |
US5968748A (en) * | 1998-03-26 | 1999-10-19 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of human HER-2 expression |
CA2337422C (fr) | 1998-08-18 | 2010-11-02 | The Regents Of The University Of California | Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r |
US6297258B1 (en) * | 1998-09-29 | 2001-10-02 | American Cyanamid Company | Substituted 3-cyanoquinolines |
US6288082B1 (en) * | 1998-09-29 | 2001-09-11 | American Cyanamid Company | Substituted 3-cyanoquinolines |
AU3281600A (en) | 1999-02-27 | 2000-09-21 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | 4-amino-quinazoline and quinoline derivatives having an inhibitory effect on signal transsduction mediated by tyrosine kinases |
AU778588B2 (en) * | 1999-10-19 | 2004-12-09 | Merck Sharp & Dohme Corp. | Tyrosine kinase inhibitors |
US6627634B2 (en) | 2000-04-08 | 2003-09-30 | Boehringer Ingelheim Pharma Kg | Bicyclic heterocycles, pharmaceutical compositions containing them, their use, and processes for preparing them |
ES2280375T3 (es) | 2000-04-08 | 2007-09-16 | BOEHRINGER INGELHEIM PHARMA GMBH & CO.KG | Heterociclos biciclicos, medicamentos que contienen estos compuestos, su uso y procedimiento para su preparacion. |
-
1999
- 1999-08-17 CA CA2337422A patent/CA2337422C/fr not_active Expired - Fee Related
- 1999-08-17 CN CNB998095745A patent/CN1184961C/zh not_active Expired - Fee Related
- 1999-08-17 US US09/375,597 patent/US6270747B1/en not_active Expired - Lifetime
- 1999-08-17 RS YU13201A patent/RS50273B/sr unknown
- 1999-08-17 SK SK216-2001A patent/SK287805B6/sk not_active IP Right Cessation
- 1999-08-17 NZ NZ509271A patent/NZ509271A/en not_active IP Right Cessation
- 1999-08-17 CZ CZ2001584A patent/CZ2001584A3/cs unknown
- 1999-08-17 PL PL351765A patent/PL200940B1/pl not_active IP Right Cessation
- 1999-08-17 WO PCT/US1999/018696 patent/WO2000010588A2/fr active IP Right Grant
- 1999-08-17 DK DK99943729T patent/DK1119349T3/da active
- 1999-08-17 AU AU56766/99A patent/AU760766B2/en not_active Ceased
- 1999-08-17 TR TR2001/00560T patent/TR200100560T2/xx unknown
- 1999-08-17 TW TW088114306A patent/TWI250019B/zh not_active IP Right Cessation
- 1999-08-17 HU HU0103894A patent/HUP0103894A3/hu unknown
- 1999-08-17 AT AT99943729T patent/ATE399541T1/de active
- 1999-08-17 EA EA200100136A patent/EA004316B1/ru not_active IP Right Cessation
- 1999-08-17 JP JP2000565908A patent/JP2002539072A/ja active Pending
- 1999-08-17 KR KR1020017002114A patent/KR100609646B1/ko not_active IP Right Cessation
- 1999-08-17 SI SI9931016T patent/SI1119349T1/sl unknown
- 1999-08-17 UA UA2001021134A patent/UA73722C2/uk unknown
- 1999-08-17 EP EP08009498A patent/EP1970058A1/fr not_active Withdrawn
- 1999-08-17 IL IL14085599A patent/IL140855A0/xx active IP Right Grant
- 1999-08-17 ID IDW20010322A patent/ID27345A/id unknown
- 1999-08-17 BR BR9912672-9A patent/BR9912672A/pt not_active IP Right Cessation
- 1999-08-17 EP EP99943729A patent/EP1119349B1/fr not_active Expired - Lifetime
- 1999-08-17 DE DE69939022T patent/DE69939022D1/de not_active Expired - Lifetime
- 1999-08-17 EE EEP200100097A patent/EE200100097A/xx unknown
- 1999-08-17 ES ES99943729T patent/ES2310047T3/es not_active Expired - Lifetime
- 1999-08-17 PT PT99943729T patent/PT1119349E/pt unknown
- 1999-08-18 MY MYPI99003537A patent/MY126490A/en unknown
- 1999-08-18 CO CO99052263A patent/CO5130028A1/es unknown
- 1999-08-19 AR ARP990104149A patent/AR020220A1/es not_active Application Discontinuation
-
2001
- 2001-01-10 IL IL140855A patent/IL140855A/en not_active IP Right Cessation
- 2001-02-14 NO NO20010749A patent/NO327000B1/no not_active IP Right Cessation
- 2001-02-16 HR HR20010119A patent/HRP20010119B1/xx not_active IP Right Cessation
- 2001-02-26 US US09/794,232 patent/US6566324B2/en not_active Expired - Lifetime
- 2001-05-24 US US09/865,239 patent/US6551989B2/en not_active Expired - Lifetime
- 2001-09-27 HK HK01106832A patent/HK1036219A1/xx not_active IP Right Cessation
-
2003
- 2003-02-07 US US10/359,982 patent/US8048844B1/en not_active Expired - Fee Related
- 2003-02-07 US US10/359,932 patent/US7358222B2/en not_active Expired - Fee Related
-
2008
- 2008-02-27 US US12/038,718 patent/US8071074B2/en not_active Expired - Fee Related
- 2008-09-26 CY CY20081101062T patent/CY1110370T1/el unknown
-
2009
- 2009-05-22 JP JP2009123658A patent/JP2009221212A/ja active Pending
-
2010
- 2010-07-21 PH PH12010000215A patent/PH12010000215A1/en unknown
- 2010-07-21 PH PH12010000216A patent/PH12010000216A1/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0157284A2 (fr) * | 1984-04-02 | 1985-10-09 | CAMILLO CORVI S.p.A. | Ester théophylline-7-acide acétique de D,L-trans-sobrérol ayant une activité mucosécrétolytique-fluidifiant et antibronchospastique, procédé pour sa préparaton et ses compositions pharmaceutiques |
WO1990003374A1 (fr) * | 1988-09-28 | 1990-04-05 | The Upjohn Company | 1,4-dihydrothionapthoquinone et congeneres heterocycliques qui inhibent les enzymes lipoxygenase |
WO1997019065A1 (fr) * | 1995-11-20 | 1997-05-29 | Celltech Therapeutics Limited | 2-anilinopyrimidines substituees utiles en tant qu'inhibiteurs de proteine kinase |
WO1999032121A1 (fr) * | 1997-12-19 | 1999-07-01 | Smithkline Beecham Corporation | .0mposes d'imidazole heteroaryle substitues, leurs compositions et usages pharmaceutiques |
Non-Patent Citations (18)
Title |
---|
AMISHIMA, M. ET AL: "Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway" AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 157, no. 6, June 1998 (1998-06), pages 1907-1912, XP000910438 * |
BLYTH, D.I. ET AL: "Induction, duration and resolution of airway goblet cell hyperplasia in a murine model of atopic asthma: effect of concurrent infection with respiratory syncytial virus and response to dexamethasone" AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, vol. 19, no. 1, July 1998 (1998-07), pages 38-54, XP000974408 * |
CRESSMAN, V.L. ET AL: "The relationship of chronic mucin secretion to airway disease in normal and CTFR-deficient mice" AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, vol. 19, no. 6, 1998, pages 853-866, XP000974409 * |
GOLDSTEIN, N.I. ET AL: "Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model" CLINICAL CANCER RESEARCH, vol. 1, 1995, pages 1311-1318, XP000891451 cited in the application * |
GRANDIS, J.R. ET AL: "Inhibition of epidermal growth factor receptor gene expression and function decreases proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells" ONCOGENE, vol. 15, no. 4, 24 July 1997 (1997-07-24), pages 409-416, XP000910341 * |
GUZMAN, K. ET AL: "Epidermal growth factor regulates expression of the mucous phenotype of rat tracheal epithelial cells" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 217, no. 2, 14 December 1995 (1995-12-14), pages 412-418, XP000907100 * |
KAWAMOTO, T. ET AL: "Growth stimulation of A431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonalantibody" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 80, March 1983 (1983-03), pages 1337-1341, XP000891494 cited in the application * |
KUMAR, R.K. ET AL: "Cooperative interaction of autocrine and paracrine mitogens for airway epithelial cells" CELL BIOLOGY AND TOXICOLOGY, vol. 14, no. 4, August 1998 (1998-08), pages 293-299, XP000909220 * |
LORIMER, I.A. ET AL: "Immunotoxins that target an oncogenic mutant epidermal growth factor receptor expressed in human tumors" CLINICAL CANCER RESEARCH, vol. 1, 1995, pages 859-864, XP000891452 cited in the application * |
LOU, Y.-P. ET AL : "Platelet-activating factor induces goblet cell hyperplasia and mucin gene expression in airways" RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 157, no. 6, June 1998 (1998-06), pages 1927-1934, XP000974347 cited in the application * |
LUNDGREN, J.D. ET AL: "Dexamethasone reduces rat tracheal goblet cell hyperplasia produced by human neutrophil products" EXPERIMENTAL LUNG RESEARCH, vol. 14, no. 6, 1998, pages 853-863, XP000974414 * |
NYCE, J.W. ET AL: "DNA antisense therapy for asthma in an animal model" NATURE, vol. 385, 20 February 1997 (1997-02-20), pages 721-725, XP000891493 cited in the application * |
SAUMA, S. ET AL: "Colon goblet cells lose proliferative response to TGFalpha as they differentiate" INTENATIONAL JOURNAL OF CANCER, vol. 61, no. 6, 1995, pages 848-853, XP000972763 * |
SCHMIDT, M. ET AL: "Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF.alpha" BRITISH JOURNAL OF CANCER, vol. 74, no. 6, 1996, pages 853-862, XP000893031 cited in the application * |
See also references of EP1119349A2 * |
STRAWN AND SHAWVER: "Tyrosine kinases in disease: overview of kinase inhibitors as therapeutic agents and current drugs in clinical trials" EXPERT OPINION ON INVESTIGATIONAL DRUGS, vol. 7, no. 4, April 1998 (1998-04), pages 553-573, XP000893015 cited in the application * |
TAKEYAMA, K. ET AL: "Epidermal growth factor system regulates mucin production in airways" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 96, no. 6, 16 March 1999 (1999-03-16), pages 3081-3086, XP000906985 * |
TAKEYAMA, K. ET AL: "Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils" JOURNAL OF IMMUNOLOGY, vol. 164, no. 3, 1 February 2000 (2000-02-01), pages 1546-1552, XP000906999 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6551989B2 (en) | 1998-08-18 | 2003-04-22 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US6566324B2 (en) | 1998-08-18 | 2003-05-20 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US6846799B1 (en) | 1998-08-18 | 2005-01-25 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US7354894B2 (en) | 1998-08-18 | 2008-04-08 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US7358222B2 (en) | 1998-08-18 | 2008-04-15 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US7531500B2 (en) | 1998-08-18 | 2009-05-12 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US7700547B2 (en) | 1998-08-18 | 2010-04-20 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US8048844B1 (en) | 1998-08-18 | 2011-11-01 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
US8071074B2 (en) | 1998-08-18 | 2011-12-06 | The Regents Of The University Of California | Preventing airway mucus production by administration of EGF-R antagonists |
WO2002005842A3 (fr) * | 2000-07-14 | 2003-01-03 | Univ California | Administration d'antagonistes d'egf visant a empecher la production de mucus dans les voies respiratoires |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6566324B2 (en) | Preventing airway mucus production by administration of EGF-R antagonists | |
US7700547B2 (en) | Preventing airway mucus production by administration of EGF-R antagonists | |
JP2010065053A (ja) | Egf−rアンタゴニストの投与による気道粘液産生の防止方法 | |
MXPA01001755A (en) | Preventing airway mucus production by administration of egf-r antagonists | |
BG105158A (bg) | Предотвратяване образуването на слузести покрития(мускус) върху летателни писти с прилагане на противодействащо средство egf-r |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: P-132/01 Country of ref document: YU Ref document number: 99809574.5 Country of ref document: CN |
|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/5-5/5, DRAWINGS, REPLACED BY NEW PAGES 1/4-4/4; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 509271 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 140855 Country of ref document: IL |
|
ENP | Entry into the national phase |
Ref document number: 2337422 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 1999 105158 Country of ref document: BG Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1200100057 Country of ref document: VN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001/00479 Country of ref document: ZA Ref document number: 200100479 Country of ref document: ZA |
|
WWE | Wipo information: entry into national phase |
Ref document number: IN/PCT/2001/141/CHE Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2162001 Country of ref document: SK |
|
WWE | Wipo information: entry into national phase |
Ref document number: PV2001-584 Country of ref document: CZ Ref document number: 56766/99 Country of ref document: AU Ref document number: 200100136 Country of ref document: EA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001/00560 Country of ref document: TR |
|
ENP | Entry into the national phase |
Ref document number: 20010128 Country of ref document: UZ Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: P20010119A Country of ref document: HR Ref document number: PA/a/2001/001755 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020017002114 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999943729 Country of ref document: EP |
|
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1999943729 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1020017002114 Country of ref document: KR |
|
WWP | Wipo information: published in national office |
Ref document number: PV2001-584 Country of ref document: CZ |
|
WWG | Wipo information: grant in national office |
Ref document number: 56766/99 Country of ref document: AU |
|
WWG | Wipo information: grant in national office |
Ref document number: 1020017002114 Country of ref document: KR |